Journal of Experimental Botany, Vol. 72, No. 2 pp. 184–198, 2021 doi:10.1093/jxb/eraa444 Advance Access Publication 25 September 2020 This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) DARWIN REVIEW Blocking intruders: inducible physico-chemical barriers against plant vascular wilt pathogens Anurag Kashyap1, , Marc Planas-Marquès1, , Montserrat Capellades1, Marc Valls1,2, and Núria S. Coll1,*, 1 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Spain 2 Genetics Department, Universitat de Barcelona, Barcelona, Spain * Correspondence: nuria.sanchez-coll@cragenomica.es Received 29 July 2020; Editorial decision 11 September 2020; Accepted 16 September 2020 Editor: Donald Ort, University of Illinois, USA Abstract Xylem vascular wilt pathogens cause devastating diseases in plants. Proliferation of these pathogens in the xylem causes massive disruption of water and mineral transport, resulting in severe wilting and death of the infected plants. Upon reaching the xylem vascular tissue, these pathogens multiply profusely, spreading vertically within the xylem sap, and horizontally between vessels and to the surrounding tissues. Plant resistance to these pathogens is very complex. One of the most effective defense responses in resistant plants is the formation of physico-chemical bar- riers in the xylem tissue. Vertical spread within the vessel lumen is restricted by structural barriers, namely, tyloses and gels. Horizontal spread to the apoplast and surrounding healthy vessels and tissues is prevented by vascular coating of the colonized vessels with lignin and suberin. Both vertical and horizontal barriers compartmentalize the pathogen at the infection site and contribute to their elimination. Induction of these defenses are tightly coordinated, both temporally and spatially, to avoid detrimental consequences such as cavitation and embolism. We discuss cur- rent knowledge on mechanisms underlying plant-inducible structural barriers against major xylem-colonizing patho- gens. This knowledge may be applied to engineer metabolic pathways of vascular coating compounds in specific cells, to produce plants resistant towards xylem colonizers. Keywords: Gels, inducible defenses, lignin, physico-chemical barriers, plant-pathogen interactions, structural defenses, suberin, tyloses, vascular pathogens, wilt Introduction The plant immune system has been shaped by hundreds of 2013). Despite the xylem being nutritionally poor in com- millions of years of interactions with microbial pathogens that parison with other plant tissues, a select group of pathogens are constantly trying to evade or overcome host plant defense comprising a few genera of bacteria, fungi and oomycetes, have reactions. Pathogens that colonize the xylem - a vital compo- evolved to target this plant tissue as a niche for colonization. nent for transporting water and minerals from the roots to the Proliferation of these wilt pathogens causes massive disrup- aerial parts - are particularly pernicious, causing major destruc- tion in the xylem vessels through the production of toxins, tion in numerous host plants worldwide (Yadeta and Thomma, exopolysaccharides, hyphal mass, conidia and other pathogen © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Inducible barriers against plant vascular wilt pathogens | 185 propagules that cause destruction as well as physical occlusion proteins (NLRs). ETI is commonly known as a stronger im- of xylem elements, resulting in severe wilting and plant death mune response, while PTI offers durable and broad-spectrum (Zaini et al., 2018). resistance (Katagiri and Tsuda, 2010). Yet, these two layers of immunity operate synergistically, converging on several down- Invasion strategies used by vascular wilt pathogens stream responses including the oxidative burst, activation of kinase signaling cascades, expression of defense-related genes Vascular plant pathogens utilize diverse strategies to get ac- and accumulation of physico-chemical barriers (Thomma et al., cess into the vessels. Vascular wilt pathogenic fungi (Fusarium 2011; Ngou et al., 2020, Preprint;Yuan et al., 2020, Preprint). oxysporum, Verticillium albo-atrum, Verticiullium dahliae, Ceratocystis Although PTI and ETI function in the interactions of fimbriata, Ophiostoma novo-ulmi), oomycetes (Pythium spp) or plants with vascular pathogens, there are very few cases the soil-borne bacterium Ralstonia solanacearum invade plant where these processes have been analyzed specifically in the roots and advance inter- or intracellularly through the root xylem and surrounding tissues of infected plants. Several re- cortex to reach the xylem, where they proliferate and spread cent studies have shown that PTI can be triggered in plants systemically to aerial plant parts (Bae et al., 2015). In order to by treatment or over-expression of conserved molecular pat- get access into the vasculature, these pathogens generally target terns present in wilt pathogens. For example, an extracellular root extremities and the junction between primary and lateral polysaccharide from the wilt bacterium R. solanacearum has roots where the epidermal barrier may be compromised, and been shown to act as a PTI elicitor in resistant tomato, leading the endodermis as well as Casparian strip are either not fully to induced expression of defense response genes (Milling differentiated or reoriented by outgrowth of lateral roots (Vasse et al., 2011; Prakasha et al., 2017). Furthermore, in Nicotiana et al., 1995; Álvarez et al., 2010). benthamiana and tomato, PTI is triggered upon perception In contrast to these root invaders, many vascular bacteria, of the COLD SHOCK PROTEIN 22 (csp22) peptide from such as Xylella fastidiosa, are directly inoculated into the xylem R. solanacearum by the PRR COLD SHOCK PROTEIN by insect vectors that feed on the plant host (Wang et al., 2017). RECEPTOR (CORE; Wang et al., 2016). In accordance, Furthermore, a few species of vascular bacteria reach the xylem treatment of tomato with csp22 conferred increased resistance tissues via natural plant openings in the aerial parts of the to R. solanacearum in tomato plants. Additionally, transgenic plant, such as leaf hydathodes (Xanthomonas oryzae pv. Oryzae; Arabidopsis thaliana plants expressing the tomato csp22 receptor Pradhan et al., 2012), flower nectarthodes (Erwinia amylovora; (SlCORE) gained the ability to respond to csp22 and were Bubán et al., 2003) or stem lenticels (Pseudomonas syringae pv. more resistant to R. solanacearum infection (Wei et al., 2018). actinidiae; Renzi et al., 2012). These bacteria multiply in inter- Similarly, V. dahliae, possesses two cellulose-degrading glyco- cellular spaces before eventually colonizing the xylem vessels. side hydrolase family 12 (GH12) proteins, namely VdEG1 and Moreover, there are vascular wilt pathogens which are trans- VdEG3, which in N. benthamiana are recognized by the PRR mitted through graft-infected rootstock/scion, as has been complexes LRR-RLP (Leucine Rich Repeat-Receptor- reported for the fungus Ceratocystis fagacearum on oak trees Like Protein)/SOBIR1/BAK1 (SUPPRESSOR OF BAK1- (Blaedow and Juzwik, 2010), or the bacterium X. fastidiosa INTERACTING RECEPTOR-LIKE KINASE 1-1/ on pecan (Sanderlin and Melanson, 2006). Another method BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED of infection of vascular wilt pathogens occurs through the RECEPTOR KINASE) and LRR-RLKs (Receptor-Like use of infected mother plants as a source for clonal propa- Kinase)/BAK1, respectively, thereby triggering PTI responses gation. This affects many plant species cultivated in nurseries, (Gui et al., 2017). such as strawberry plants infected with Fusarium oxysporum Concomitantly, some bacterial wilt pathogens have evolved f. sp. fragariae (Pastrana et al., 2019), olive plants infected with modifications in the sequences of their conserved patterns, so V. dahliae (Morello et al., 2016), and it is also common in grape- that they are no longer recognizable by cognate PRRs. This vine mother plants that can harbor various vascular wilt fungi occurs in X. fastidiosa, which contains a lipopolysaccharide (Gramaje et al., 2018). featuring a masking motif that evades recognition by grape- vine (Rapicavoli et al., 2018), or flagellin from R. solanacearum, Immunity against vascular wilt pathogens with a polymorphic sequence that avoids perception by many host plants (Wei et al., 2020). Plants are protected against pathogens by a well-orchestrated An interesting emerging concept is that roots may be rela- immune system (Jones and Dangl, 2006). Pattern-triggered im- tively insensitive to most conserved molecular patterns, which munity (PTI) is initiated upon recognition of conserved mi- may prevent mounting defense responses against commensal crobial features at the plasma membrane by pattern-recognition organisms in an environment such as the soil, full of microbes. receptors (PRRs) in the plant. Effector-triggered immunity Underlying this phenomenon, it has been recently shown that (ETI) is activated by recognition of pathogen-secreted ef- root PTI is only activated upon local tissue damage (Zhou fectors via intracellular nucleotide-binding leucine-rich repeat et al., 2020). This work convincingly shows how localized cell 186 | Kashyap et al. death upregulates PRR expression in the neighboring cells, 2018), and watermelon (Lambel et al., 2014). QTLs termed leading to restricted responsiveness to conserved molecular RESISTANCE TO FUSARIUM (RFO1-RFO7) have been patterns of root invaders (Zhou et al., 2020). identified in Arabidopsis that provide broad spectrum im- ETI responses against vascular pathogens have also been munity to multiple formae speciales of F. oxysporum (Chen et al., documented in the literature. For instance, in the interaction 2014). RFO1 was found to encode a wall-associated kinase- between tomato and the vascular wilt fungus F. oxysporum f. sp. like kinase 22 (WAKL22) and RFO2 encodes a receptor-like lycopersici, several R protein-effector pairs have been identi- protein (Diener and Ausubel, 2005). Likewise, resistance to fied. The NLR I2 perceives the Avr2 effector; I, a receptor-like Verticillium correlates with mapping of QTLs in several hosts protein with an extracellular LRR domain (LRR-RLP) per- such as strawberry (Antanaviciute et al., 2015) and cotton ceives Avr1; and I3, a S-receptor-like kinase (SRLK) perceives (Palanga et al., 2017). A major QTL conferring resistance to Avr3 (Houterman et al., 2009; Catanzariti et al., 2015, 2017). X. fastidiosa was identified in a Vitis arizonica linkage map and Interestingly, the I2 gene is primarily expressed in the xylem named as ‘Pierce’s disease resistance 1’ (PdR1), which has been vascular tissue, probably partly explaining why the fungus introgressed into commercial cultivars (Krivanek et al., 2006). reaches the vasculature before being contained in an incom- However, the majority of available QTL mapping data for wilt patible interaction (van der Does et al., 2019; Mes et al., 2000). resistance traits lack resolution. This may be partly due to the Similarly, effector AvrFom2 from F. oxysporum f. sp. melonis is high sensitivity of wilt development to temperature variation, perceived by the NLR Fom-2 in melon (Joobeur et al., 2004). and also due to the fact that wilt resistance comprises a complex On the other hand, the effector Ave1 from V. albo-atrum induces array of multilayered mechanisms involving the coordinated resistance in several plant species, mediated by the receptor-like action of different plant tissues or cell types (Bani et al., 2018). protein Ve1 (de Jonge et al., 2012; Song et al., 2018). Importantly, this extremely complex polygenic and quantita- With respect to bacterial vascular wilt pathogens, the tive resistance underscores the structural physico-chemical de- RRS1-R/RPS4 (Resistance to Ralstonia solanacearum fense mechanisms induced by vascular pathogens in resistant 1-Recessive/ Resistant to Pseudomonas syringae 4) NLR pair plants. in Arabidopsis mediates immunity against R. solanacearum carrying the PopP2 effector (Deslandes et al., 2002; Le Roux Inducible structural barriers to restrict the spread of et al., 2015; Sarris et al., 2015). The rice LRR-RLK protein vascular wilt pathogens Xa21 confers broad-spectrum immunity to X. oryzae pv. oryzae, mediated through recognition of the small protein Inducible structural barriers formed at and around the vas- Ax21 (Park and Ronald, 2012). In addition, the rice NLR culature upon colonization constitute one of the most im- Xo1 recognizes the transcription activator-like effector Tal2H portant defense components against wilt diseases. If a pathogen from X. oryzae pvs. oryzicola and oryzae (Triplett et al., 2016). manages to reach the xylem, this transport system becomes an Similarly, the NLR Bs4 in tomato perceives the avirulence excellent channel of inoculum dissemination throughout the protein AvrBs4 from Xanthomonas campestris pv. vesicatoria plant. As a consequence, plants have evolved effective structural (Schornack et al., 2004). Furthermore, the Arabidopsis NLR defense mechanisms to prevent vessel colonization or move- ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) recognizes ment between vessels once vascular colonization has occurred the X. campestris effector AvrAC/XopAC by forming a com- (Beckman and Roberts, 1995) Timely formation of these plex with PBL2 (PROBABLE SERINE/THREONINE- physico-chemical vascular barriers early upon pathogen per- PROTEIN KINASE 2), which acts as a decoy, and RKS1 ception can lead to confinement of the vascular pathogen at (RESISTANCE-RELATED kinase 1), a pseudokinase (Wang the infected vessel, avoiding the spread of wilt diseases (Robb et al., 2015). et al., 2007; Zaini et al., 2018; Planas-Marquès et al., 2019). Besides individual examples of R genes, quantitative traits Some of these structural reinforcements induced by patho- have been shown to underscore immunity against xylem gens were already reported in classic botanical studies in the colonizers in many different hosts. Stable resistance towards 19th century (Zimmermann, 1979). Moreover, during the R. solanacearum in tomato is controlled by two major quan- 1980s and 1990s, they were intensely studied from anatomical titative trait loci (QTLs), namely Bwr-6 and Bwr-12, located and biochemical points of view, as they were recognized as in chromosomes six and twelve, respectively, and three minor important components of defense reactions (Newcombe and QTLs, Bwr-3, Bwr-4 and Bwr-8 (Thoquet et al., 1996; Mangin Robb, 1988; Nicholson, 1992; Niemann, 1994). In trees, the et al., 1999; Wang et al., 2000, 2013; Carmeille et al., 2006). sequential response to confine fungal pathogen progression or Some of these loci have been shown to be strain and/or tissue damage at the site of infection or injury was initially environment-specific, and involve an extremely complex gen- explained by the CODIT model (compartmentalization of etic basis of resistance. Similarly, QTLs for Fusarium wilt re- decay in trees; Shigo and Marx, 1977). The model described sistance have been mapped in several hosts such as chickpea four “walls” or barriers pre-formed or formed in response to (Sabbavarapu et al., 2013), cotton (Ulloa et al., 2013;Wang et al., wounding, that restrict pathogen colonization. In particular, Inducible barriers against plant vascular wilt pathogens | 187 wall 1 defined vessel plugging structures formed as a response into xylem vessels through its pits (Fig. 1; Bonsen and Kucera, to vascular wilt pathogen invasion. 1990). Tyloses are considered inducible defense structures However, most of the research in the field of plant-pathogen against xylem intruders because their formation can prevent interactions started refocusing during the 1990s when spreading of the pathogen and also protect healthy parts of the Arabidopsis gained momentum as a model species. This led plant by blocking the infected vessels (Leśniewska et al., 2017). to the identification of striking similarities between plant and However, formation of tyloses is not only linked to pathogen animal immune systems, the type III secretion system in bac- attack, as they can be induced by several other environmental teria being discovered, and avr-R gene pairs being identified stimuli, such as pruning, wounding, flooding and frost (Davison as a corollary of Flor’s gene-for-gene model (Nishimura and and Tay, 1985; Cochard and Tyree, 1990; Sun et al., 2007; De Dangl, 2010). We now think it is time to revisit the role of Micco et al., 2016). The process of tylosis formation is tightly inducible structural defenses in plant-pathogen interactions. controlled and has certain commonalities with regular cell These defenses are extremely important to block progression of enlargement (Bishop and Cooper, 1984). Hormones such as the devastating vascular pathogens, and we are far from under- auxin, ethylene and jasmonate seem to have a prominent role standing how these types of defense mechanisms are controlled. in the formation of these structures (Vander Molen et al., 1987; In this review, we summarize how inducible vascular struc- Leśniewska et al., 2017). tures compartmentalize the vascular wilt pathogens leading The formation of tyloses in response to pathogen attack to resistance. For this, we classify inducible structural barriers has been extensively reported (Table 1; Cooper and Williams, using a bi-dimensional perspective. We define a vertical and a 2004; Sun et al., 2013; Ferreira et al., 2017; Rioux et al., 2018). horizontal component of resistance as those structures that re- Restricted formation of tyloses has been observed and specif- strict the vertical and horizontal movement of the pathogen, ically induced in the infected vessels of resistant tomato and respectively, within the xylem vascular tissue (Fig. 1, Table 1). potato varieties, effectively restricting R. solanacearum to the Furthermore, we review current knowledge on mechanisms infected vascular bundles (Grimault et al., 1994; Ferreira et al., underlying plant-inducible structural defenses against major 2017). Such specific induction was not observed in suscep- xylem-colonizing pathogens, highlighting their correlation tible tomato cultivars infected with R. solanacearum, where the with biochemical changes and genetic interactions. Finally, we formation of tyloses appeared delayed and less focused, with discuss future perspectives in the study of inducible vascular numerous non-colonized vessels occluded by tyloses, and structural defenses, taking into account recent technological ad- pathogen growth unrestricted (Grimault et al., 1994). Tylosis vances and how this may be translated into increasing resistance formation in resistant tomato cultivars has also been observed to vascular wilt pathogens in the field. Such physico-chemical upon inoculation with the pathogenic fungus F. oxysporum defense responses are key traits desired for effective manage- f. sp. lycopersici and V. albo-atrum (Hutson and Smith, 1980). ment of vascular wilt pathogens (Ferreira et al., 2017). Similarly, in a banana cultivar resistant to race 1 of F. oxysporum f. sp. cubense, tylosis initially appeared as early as within two Vertical restriction of vascular colonization days post-inoculation in the lumen of xylem vessels of the root (Vander Molen et al., 1987). Once they reach the xylem, different lumen occlusions con- In addition to acting as structural barriers, tyloses can act stitute vertical barriers to the anti-gravitational movement of as storage organs of antimicrobial compounds. Although they vascular pathogens. Vascular occlusion is an effective means of are predominantly composed of pectic substances (Rioux slowing down vertical progression of the pathogen, or even et al., 1998; Eynck et al., 2009), fungicidal compounds such as confining it to the infection site, preventing systemic infection. elemental sulfur have been detected in tyloses of tomato lines The most prominent of these occlusions are tyloses and gels, resistant to V. albo-atrum, which was thought to inhibit spore which we will review in this section, focusing on the aspects germination (Williams et al., 2002). In addition, a fully devel- related to plant defense. As an important defense strategy oped tylosis also develops a lignified and/or suberized cell wall, by vascular pathogens, mechanisms to evade or subvert ver- which may protect the exposed cell from the xylem pathogen tical occlusions and secure colonization have evolved on the (Pouzoulet et al., 2013). pathogen side, as part of the evolutionary arms race. Inhibition However, the formation of tyloses is not always linked to of vertical occlusions has been reported for various wilt fungi, resistance. For instance, a tomato variety susceptible to V. albo- although the mechanisms for this virulence mechanism are atrum formed a few miniature tyloses in response to infec- not fully understood (Beckman and Roberts, 1995; Pouzoulet tion that the fungal hyphae were able to easily surpass within et al., 2017). the vessel (Tjamos and Smith, 1975). More important than the quantity or frequency of tyloses formed, the successful re- Formation of tyloses striction of a vascular pathogen depends on synchronization and specificity of tylose production to pathogen-colonized Tyloses (singular tylosis) are balloon-like overgrowths of the vessels (Bishop and Cooper, 1984). Sun et al., (2013) reported protoplast of adjacent living parenchyma cells that protrude tyloses as the predominant type of occlusion that formed in 188 | Kashyap et al. Fig. 1. The two dimensions of plant physico-chemical barriers induced against xylem vascular wilt pathogens. To counter invasion by xylem vascular wilt pathogens, resistant plants induce two-dimensional physico-chemical defenses that restrict vertical and horizontal movement of the pathogen. Vertical spread within the vessel lumen is mainly restricted by tyloses and gels (left). In contrast, horizontal spread of the pathogen to surrounding healthy vessels is prevented by reinforcement of the walls of colonized vessels (V) and the surrounding xylem parenchyma (XP) and tracheids (T), through vascular coating with mainly lignin and suberin (shown as a green color in the diagram). Synchronized formation of vertical and horizontal barriers early after pathogen invasion results in compartmentalization of the pathogen inoculum at the site of infection, thereby preventing wilt, and constitute a major component of resistance. To visualize tyloses and vascular coating (panels below), tomato root cross-sections were obtained after R. solanacearum soil soak inoculation, and fixed in 70% ethanol. For tyloses, cross-sections were stained with 0.1% toluidine blue and observed using a Leica DM6B-Z microscope under bright field conditions, and images were recorded through a MC190-HD-0518131623 camera. To visualize phenolic vascular coating, the cross-sections were illuminated by UV using a Leica DM6B-Z microscope, and the auto-florescence emitted from phenolic deposits was observed using a HC PL FLUOTAR objective. Images were captured using a Leica-DFC9000GT-VSC07341 camera. In the left panel, the arrow points towards R. solanacearum-induced formation of tyloses inside vessel lumen, which appear pink to violet color upon staining with 0.1% toluidine blue. In the right panel, the arrow points towards R. solanacearum-induced auto-fluorescence emitted from phenolics, deposited in the walls of vessels and the surrounding tracheids and parenchyma cells. Scale bar=120 µm. grapevines with differing resistance to X. fastidiosa. Excessive are key (Sun et al., 2013). Interestingly, the anatomy of xylem tylosis formation in response to X. fastidiosa infection in the vessels also plays a role in augmenting pathogen compartmen- susceptible grapevine cultivar led to heavy blockage of vessels talization by tyloses. Cultivars of grapevine having larger vessel and development of wilting symptoms, and did not signifi- size are known to be susceptible to vascular wilt pathogens cantly affect pathogen spread. In contrast, in resistant grape- such as X. fastidiosa, Eutypa lata, Phaeoacremonium aleophilum, vines, tylosis development was specific and mainly limited to Phaeomoniella chlamydospora, Diplodia seriata, and Neofusicoccum a few internodes close to the point of inoculation, impacting parvum (Pouzoulet et al., 2017, 2019, 2020; Deyett et al., 2019). less of the vessels and indicating that timing and localization Likewise, Dutch elm cultivars having larger vessel diameters Inducible barriers against plant vascular wilt pathogens | 189 Table 1. List of plant pathosystems in which (I) vertical or (II) horizontal restriction of pathogen movement inside the plant has been shown. I. Vertical restriction Structure Host Pathogen Reference Tylose formation Banana Fusarium oxysporum f.sp. cubense (Vander Molen et al., 1987) Butternut Ophiognomonia clavigignenti-juglandacearum (Rioux et al., 2018) Cotton Fusarium oxysporum f. sp. vasinfectum (Shi et al., 1991) Cucurbits Fusarium oxysporum f. sp. melonis (Seo and Kim, 2017) Elm Ophiostoma novo-ulmi (Plichta et al., 2016) Grapevine Xylella fastidiosa (Sun et al., 2013) Phaeomoniella chlamydospora (Pouzoulet et al., 2013) Potato Rasltonia solanacearum (Ferreira et al., 2017) Tomato Rasltonia solanacearum (Grimault et al., 1994) Verticillium albo-atrum (Hutson and Smith, 1980) Verticillium dahliae (Tjamos and Smith, 1975) Fusarium oxysporum f. sp. lycopersici (Hutson and Smith, 1980) Gel deposition Banana Fusarium oxysporum f.sp. cubense (Vander Molen et al., 1987) Butternut Ophiognomonia clavigignenti-juglandacearum (Rioux et al., 2018) Carnation Fusarium oxysporum f.sp. dianthi (Baayen and Elgersma, 1985) Elm Ophiostoma novo-ulmi (Plichta et al., 2016) Grapevine Xylella fastidiosa (Sun et al., 2013) Pea Fusarium oxysporum f. sp. pisi (Bishop and Cooper, 1983) Plane tree Ceratocystis fimbriata f. sp platani (Clérivet et al., 2000) Tomato Rasltonia solanacearum (Grimault et al., 1994; Kim et al., 2016) Verticillium albo-atrum (Hutson and Smith, 1980) Fusarium oxysporum f. sp. lycopersici (Hutson and Smith, 1980) II. Horizontal restriction Structure Host Pathogen Reference Lignin deposition Banana Fusarium oxysporum f. sp. cubense (De Ascensao and Dubery, 2000) Cotton Verticillium dahliae (Xu et al., 2011; Bu et al., 2014) Dutch elm Ophiostoma novo-ulmi (Martin et al., 2007) Flax Fusarium oxysporum f. sp. lini (Galindo-González and Deyholos, 2016) Oilseed rape Verticillium longisporum (Eynck et al., 2009) Olive Xylella fastidiosa (Sabella et al., 2018) Pepper Verticillium dahliae (Novo et al., 2017) Potato Rasltonia solanacearum (Ferreira et al., 2017) Tomato Verticillium dahliae (Street et al., 1986; Hu et al., 2019) Rasltonia solanacearum (Ishihara et al., 2012; Ferreira et al., 2017) Suberin deposition Alfalfa Verticillium albo-atrum (Newcombe and Robb, 1988) Butternut Ophiognomonia clavigignenti-juglandacearum (Rioux et al., 2018) Dutch elm Ophiostoma novo-ulmi (Martín et al., 2008) Grapevine Phaeomoniella chlamydospora (Pouzoulet et al., 2013) Tomato Verticillium albo-atrum (Street et al., 1986; Robb et al., 1991) are susceptible to Ophiostoma novo-ulmi (Pouzoulet et al., 2014; Deposition of gels Venturas et al., 2014). Across the grapevine genotypes, it has been observed that the extent of P. chlamydospora compart- Deposition of electrodense material corresponding to gels or mentalization is a function of the diameter of the host xylem gums in the lumen of the xylem vessels is another feature com- vessels. Genotypes with increased number of xylem vessels monly observed during xylem invasion by vascular pathogens, above 100 µm in diameter resulted in increased infection of and acts as one of the multiple factors that contribute to in- host tissue (Pouzoulet et al., 2020). Though numerous tyloses duced structural defense (Vander Molen et al., 1977). Besides are formed in large vessels of susceptible grapevine cultivars in its role in immunity, these occluding structures also form in re- response to the wilt pathogen P. chlamydospora, the compart- sponse to other stimuli such as wounding or aging (Ratnayake mentalization process is not as efficient as in narrow diameter et al., 2013). vessels, due to the presence of large escape routes (Pouzoulet Gels are commonly secreted by xylem parenchyma cells and et al., 2017). they are transported across pit membranes into vessel elements 190 | Kashyap et al. (Fig. 1; Bishop and Cooper, 1984). Tyloses have also been ob- pathogens, resistant plants with altered composition and struc- served to secrete gels into the lumen of vessels, thereby multi- ture of homogalacturonans (HGs) and xyloglucans (XyGs) in plying the clogging effect (Bonsen and Kucera, 1990; Rioux pit membranes have evolved; however, these compounds are et al., 1998). Gels appear fibrillar, forming thin networks of potential targets of the pathogen’s cell wall degrading enzymes. varying electron density that ultimately fill and clog the vessel Grapevine genotypes resistant to X. fastidiosa lacked fucosylated lumen (Sun et al., 2008). Gels initially appear as translucent XyGs and weakly methylesterified HGs (ME-HGs), and con- fibres arising from several places along lateral walls of vessels, tained a small amount of heavily ME-HGs. In contrast, pit and later form a continuous layer with wavy edges toward the membranes of susceptible genotypes all had substantial amounts vessel lumen. Subsequently, gels turn yellow and are inter- of fucosylated XyGs and weakly ME-HGs, but lacked heavily spersed with small particles, coinciding with vessel occlusions ME-HGs (Sun et al., 2011). (Sun et al., 2008). Although the main component of gels are In addition, reinforcement occurs at vessel walls, paren- pectic substances such as partially esterified pectic polysac- chyma cells and pit membranes, to confine the spread of vas- charides (Rioux et al., 1998; Clérivet et al., 2000), they may cular pathogens. Ultra-microscopic studies showed that the also accumulate antimicrobial compounds such as elemental pit membranes, as well as vessels walls and parenchyma cells, sulfur and phytoalexins (Cooper and Williams, 2004; Sun et al., form a conspicuously thick coating in the form of an elec- 2008). Furthermore, these gels are strengthened by deposition tron dense amorphous layer, as part of the defense response of lignin and other phenolic compounds, which make these against vascular pathogens (Street et al., 1986; Benhamou, 1995; plugs strong physical barriers (Kpemoua et al., 1996; Rioux Daayf et al., 1997; Nakaho et al., 2000; Araujo et al., 2014). Such et al., 1998). reinforcement acts to limit the horizontal movement of the Formation of vascular gels is considered an important part of pathogen from the protoxylem or the primary xylem to the resistance towards several wilt diseases (Table 1). For example, surrounding cells (Street et al., 1986; Benhamou, 1995; Daayf F. oxysporum f. sp. dianthi colonization is restricted due to the et al., 1997; Nakaho et al., 2000; Araujo et al., 2014). Besides, formation of gels in the vascular lumen of carnation plants its deposition acts as a shield against pathogen-derived metab- (Baayen and Elgersma, 1985). Moreover, in most resistant culti- olites such as toxins and enzymes, and makes water and nutri- vars these gels are often observed together with tyloses (Vander ents inaccessible for pathogens, thereby impeding their growth Molen et al., 1987; Grimault et al., 1994). In banana plants re- (Araujo et al., 2014). sistant to F. oxysporum f. sp cubense, formation of vascular oc- Importantly, the timing of synthesis of the vascular coating clusions including both gels and tyloses have been observed plays a crucial role in immunity. Even if vascular coating can (Vander Molen et al., 1977). Gel formation in the xylem lumen be observed in response to vascular pathogens in susceptible is also a trait of tomato cultivars resistant towards the vascular plants, these structures form at late time points, compared with bacterium R. solanacearum (Grimault et al., 1994). In other their induction in resistant plants (Shi et al., 1991; Daayf et al., cases, such as pea plants resistant to F. oxysporum f. sp. pisi, vas- 1997). The specific composition of vascular deposits varies de- cular gels, but not tyloses, are observed after infection (Bishop pending on the particular host-pathogen interaction. However, and Cooper, 1984). phenolics are the most important compounds, as they act as building blocks of the secondary cell wall, and they also have Horizontal restriction of vascular colonization direct antimicrobial activity (Eynck et al., 2009). Among the phenolic polymers constituting vascular coating structures, the The mechanisms mentioned above are part of host responses principal players are lignin and suberin, described in further that restrict vertical movement of vascular pathogens to detail below.We also outline the role of callose, a non-phenolic healthy regions of the host. In addition, resistant plants can compound that plays an important role in the formation of often develop a protective vascular coating upon invasion by horizontal vascular barriers in certain interactions. vascular pathogens, posing a horizontal barrier to further col- onization of adjacent healthy tissues. Vascular coating involves Deposition of lignin physico-chemical structural modifications in the cell walls of xylem tissues that result in confinement of the pathogen to the Lignin is a complex phenolic polymer that constitutes a infected vessels (Figs 1, 2A). major component of secondary cell walls in vascular plants. Xylem pits are the primary routes of vessel-to-vessel and Lignin imparts strength to secondary cell walls, being depos- vessel-to-parenchyma cell water transport. Pits are covered by ited in spaces between cellulose, hemicellulose and pectin a pit membrane, which is impermeable to particulate matter (Kang et al., 2019; Fig. 2A). The building blocks of the lignin like bacteria and other pathogens (Choat et al., 2008). For a polymer are monolignols, synthesized from phenylalanine via pathogen to achieve successful horizontal transfer into ves- the phenylpropanoid pathway, where numerous enzymes are sels, it has to either form openings in vessel walls or degrade involved (Fig. 2B). Monolignols are then transported from the pit membranes, thereby reaching the adjacent parenchyma cytosol into the apoplast, where they are polymerized to lignin cells and vessels (Nakaho et al., 2000). To avoid the breach by units by the oxidative activities of laccases and peroxidases. Inducible barriers against plant vascular wilt pathogens | 191 A Vessel Parenchyma B Secondary wall Middle lamella Plasma membrane Primary wall Shikimate Phenylalanine pathway PAL Invasion Cinnamic acid Phenylpropanoid pathogens pathway Vascular C4H p-coumaric acid C4H 4CL Caffeic acid C3H p-coumaroyl-CoA COMT HCT 4CL p-coumaroyl shikimate Suberin Lignin C3H HCT Hemi-cellulose Cellulose Caffeoyl shikimate Caffeoyl-CoA CCoAOMT 4CL Acyl lipid Feruloyl-CoA Ferulic acid Suberin Fatty acids pathway pathway CYP86A1 FAR ω-hydroxyacids Primary Alcohols Lignin pathway CCR CCR F5H p-coumaraldehyde Coniferaldehyde Sinapaldehyde Tyrosine COMT CAD CAD CAD TyDC p-coumaryl alcohol Coniferyl alcohol Sinapyl alcohol Tyramine FHT Lignin THT monomers PRX PRX PRX Feruloyl tyramine Suberin Feruloyl esters p-coumaroyl tyramine p-coumaroyl esters p-hydroxyphenyl (H) Guaiacyl (G) Syringyl (S) monomers Caffeoyl tyramine Caffeoyl esters unit unit unit PRX H LIGNIN G LIGNIN S LIGNIN SUBERIN Fig. 2. Lignin and suberin have a major role in vascular coating induced by xylem vascular wilt pathogens. A. Schematic structure of reinforced cell walls of xylem vessels and parenchyma cells in resistant plants upon infection with xylem vascular wilt pathogens. B. The phenypropanoid pathway provides precursors for both lignin and suberin biosynthesis. Phenylalanine, derived from the shikimate pathway, undergoes several enzymatic reactions as part of the phenylproanoid pathway. The resulting precursors yield the monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, which are the building blocks of lignin. In parallel, the phenypropanoid metabolites feruloyl-CoA, caffeoyl-CoA, and p-coumaroyl-CoA bifurcate into the suberin pathway. In the suberin pathway, these metabolites can be conjugated to aromatic amine compounds such as tyramine by the action of THT, or can be linked to aliphatic compounds by the action of FHT, to yield suberin monomers. Lignin and suberin monomers are then transported to the cell wall, where they are subsequently polymerized into the reinforcing matrices that constitute vascular coating structures. Abbreviations: PAL: phenylalanine ammonia–lyase; C4H: cinnamate–4–hydroxylase; C3H: coumarate 3-hydroxylase; 4CL: 4–coumarate–CoA ligase; HCT: hydroxycinnamoyl– CoA shikimate/quinate hydroxycinnamoyl transferase; COMT: caffeic acid 3-O-methyltransferase; CCOMT: caffeoyl CoA 3-O-methyltransferase; CCR: cinnamoyl CoA reductase; CAD: cinnamoyl alcohol dehydrogenase; PRX: peroxidase; CYP86A1: fatty acid cytochrome P450 oxidases; FAR: fatty acyl- CoA reductase; TyDC: tyrosine decarboxylase; FHT: feruloyl transferase; THT: tyramine hydroxycinnamoyl transferase. Lignin is a particularly important element in cell walls of reinforcements that further restrict pathogen colonization.This xylem tissue cells, not only because of its structural function, inducible deposition of lignin as a vascular coating can be an but also because it facilitates water retention in vascular bun- important component of resistance towards certain pathogens dles due to its hydrophobic nature. In addition, lignin is re- (Table 1). sistant to biodegradation and acts as a potent structural barrier Restriction of pathogen colonization by lignin has been ele- against pathogens. Although lignin is an integral part of pre- gantly shown using a non-vascular pathogen, the leaf-infecting existing structural barriers in plants, its deposition can also be bacterium Pseudomonas syringae pv tomato (Pto) on Arabidopsis induced in the xylem upon pathogen attack to form additional thaliana (Lee et al., 2019). It was shown that infection with 192 | Kashyap et al. either virulent or avirulent strains of the pathogen induced Nevertheless, the mechanisms that orchestrate timely and localized lignification at the site of pathogen attack. However, effective induction of lignin deposition in resistant plants lignin deposition was more conspicuous upon avirulent Pto upon vascular colonization remain vastly unknown. Some recognition, leading to confinement of the pathogen, and re- progress has been made using cotton plants infected with stricting hypersensitive response cell death to the infection site. V. dahliae. In this pathosystem, two proteins potentially regu- In contrast, virulent strains could overcome induced lignifica- lating V. dahliae-induced vascular lignin deposition have been tion, as the deposition in this case was milder, leading to unre- identified. GhUMC1, a copper-binding protein, is involved stricted disease progression. in resistance mediated by lignin deposition and jasmonate Interestingly, Casparian strip membrane domain proteins signaling (Zhu et al., 2018). GhUMC1 knock-down plants are (CASPs), were shown to be involved in pathogen-induced more susceptible to the pathogen, and lignin vascular coating lignin deposition (Lee et al., 2019). The Casparian strip acts is drastically reduced. On the other hand, the proline-rich as a diffusion barrier that controls the uptake of water and protein GhHyPRP1 acts as a negative regulator of defense molecules from the soil into the water-conducting tissues, and against V. dahlia, and was shown to induce lignin deposition prevents the entry of pathogens and other harmful substances. (Zhu et al., 2018). In accordance, GhHyPRP1 knock-down Both the Casparian strip and the above-mentioned pathogen- plants displayed more lignin deposition upon infection and induced lignification mechanism involve lignin-containing were more resistant to the pathogen. Interestingly, PevD1, a structures that function as anti-pathogenic physical barriers, V. dahliae secreted protein, has been shown to activate defenses serving parallel functions. In addition to lignin, suberin (dis- in cotton, triggering the expression of phenylpropanoid genes cussed in detail later) is a chief component of the Casparian and lignin accumulation (Bu et al., 2014). This may indicate strip (Doblas et al., 2017). Recent findings show that the that PevD1 acts as an avirulence effector in cotton triggering Casparian strip possesses a barrier surveillance pathway com- a defense reaction that includes lignin deposition in the vascu- prised of the receptor-like cytoplasmic kinase SCHENGEN1 lature, which may lead to pathogen confinement into infected and the LRR-RLK SCHENGEN3, hence bearing a striking vessels. It remains to be determined whether the differential resemblance to signaling pathways for perception of pathogen- lignin deposition phenotypes observed in resistant cultivars is associated molecular patterns (Alassimone et al., 2016; Fujita a direct consequence of pathogen effector recognition via its et al., 2020). These receptors in the Casparian strip domain secreted effectors, and if so, to what degree the mechanisms are interact with peptides (CASPARIAN STRIP INTEGRITY conserved between different pathosystems. Moreover, the mo- FACTORS, CIF1/2) expressed in the stele, leading to lecular players involved also need to be identified. Casparian strip formation. This spatial separation of receptor and ligand constitutes a surveillance system where interaction Deposition of suberin stops after effective sealing by the strip, but any breach in the barrier leads to re-interaction and further strengthening Suberin is a heteropolymer that deposits as a poly-lamellar (Doblas et al., 2017). structure between the plasma membrane and the cell wall, Lignin deposits seem to also play a crucial role in spa- forming a hydrophobic protective barrier (Fig. 2A). Suberin tial growth restriction of vascular pathogens. There are is deposited in specialized tissues such as root and tuber epi- several examples showing a pronounced transcriptional dermis, root endodermis, and seed coats. In addition, suberin upregulation of genes involved in lignin biosynthesis in is formed in response to several stresses such as wounding, salt resistant plants following infection with various vascular injury and pathogen attack (Dixon and Paiva, 1995; Bernards, pathogens (Table 1). This occurs, for example, in cotton after 2002). Besides providing strength to the cell wall, suberin pre- V. dahliae infection, flax after infection with F. oxysporum, vents water loss and pathogen entry by sealing off the layer of tomato after infection with R. solanacearum, or olive tree suberized cells. after X. fastidiosa infection (Xu et al., 2011; Ishihara et al., Suberin consists of a polyphenolic and a polyaliphatic domain. 2012; Galindo-González and Deyholos, 2016; Sabella et al., The polyphenolic domain is predominantly formed by esters 2018). Furthermore, there are several examples showing and amides of ferulic acid, as well as other hydroxycinnamic that resistance/tolerance to vascular pathogens is accom- acids, such as caffeic acid and p-coumaric acid (Negrel et al., panied by an increase in lignin content and enhanced cell 1995; Lashbrooke et al., 2016; Woolfson et al., 2011; Woolfson, wall lignification upon infection. This includes examples of 2018). The aliphatic domain consists of a glycerol-based fatty different pathosystems such as V. dahliae—pepper, O. novo- acid-derived polyester comprised primarily of ω-hydroxyacids, ulmi—Ulmus minor, V. longisporum—rapeseed, F. oxysporum— α, ω-dicarboxylic acids, fatty alcohols, and small amounts of banana or tomato, and R. solanacearum—potato (Street et al., hydroxycinnamic acids (mainly alkyl ferulates; Beisson et al., 1986; De Ascensao and Dubery, 2000; Martín et al., 2005; 2012). Ferulic esters composed of ferulic acid esterified to Martin et al., 2007; Eynck et al., 2009; Ferreira et al., 2017; fatty acids are considered as one of the monomers of suberin Novo et al., 2017). polymer (Negrel et al., 1995). Inducible barriers against plant vascular wilt pathogens | 193 Significant progress elucidating suberin biosynthesis has little is known about its regulation. Similar to lignin, the effect- been achieved in the last two decades using molecular gen- iveness of vascular suberization as a structural barrier against etics approaches, especially in the model species A. thaliana and horizontal colonization by vascular pathogens largely depends in potato tuber periderm (Ranathunge et al., 2011). Suberin on the spatio-temporal control of its deposition, i.e. formation shares the phenypropanoid pathway with lignin (Fig. 2B). This of suberin deposits early after pathogen detection at the site pathway provides the precursors for its polyphenolic domain, of vascular invasion. The phytohormones abscisic acid (ABA) which are used by downstream suberin-specific enzymes such and ethylene have both been shown to regulate suberization as tyramine N-feruloyltransferase (THT) and feruloyl trasferase (Soliday et al., 1978; Cottle and Kolattukudy, 1982; Barberon (FHT; Fig. 2B; Serra et al., 2010; Woolfson, 2018; Woolfson et al., 2016). Since these two hormones are involved in de- et al., 2011). Similarly, several genes involved in the aliphatic fense against various vascular pathogens, a possibility exists that metabolism of suberin have been described, such as fatty acid pathogen-induced vascular suberization correlates with an in- cytochrome P450 oxidases (CYP86A1), fatty acyl-CoA re- crease of hormone concentrations during immune responses. ductase (FARs), β-ketoacyl-CoA synthases (KCS2/Daisy and However, the mechanistic links between these hormonal path- KCS20), glycerol-3-phosphate acyltransferase5 (GPAT5), as ways and suberin biosynthesis during pathogen-triggered su- well as ATP-BINDING CASSETTE G (ABCG) genes in- berin vascular coating remain to be established. volved in the delivery of suberin monomers to the site of su- berization (ABCG2, ABCG6, and ABCG20; Beisson et al., Deposition of callose 2007; Höfer et al., 2008; Franke et al., 2009; Vishwanath et al., 2013;Yadav et al., 2014). In addition, a few upstream regulators Callose is a linear amorphous cell wall polysaccharide formed of the suberin biosynthetic pathway have been identified in by hundreds of glucose units linked by β-1,3 glucosidic bonds A. thaliana, such as the MYB transcription factors MYB41, (Stone, 2009). This homopolysaccharide is synthesized from MYB107 and SUBERMAN (MYB39; Kosma et al., 2014; uridine diphosphate glucose by callose synthases (also known Gou et al., 2017; Cohen et al., 2020). as CalS or GSL for glucan synthase-like), large multisubunit Suberin reinforcement in the xylem vascular tissue has been complexes at the plasma membrane (Ellinger and Voigt, 2014). long recognized as a potent barrier to colonization by patho- Callose is not a particularly abundant polymer in the cell wall, gens (Robb et al., 1991). There are numerous studies reporting but has very relevant regulatory roles in development, plasmo- that suberin deposition in the xylem tissue upon infection desmata function, as well as in immunity (Schneider et al., contributes to resistance (Table 1). For example, vascular de- 2016). Pathogen-induced callose deposition has been shown posits have been observed after infection with V. albo-atrum to be localized to callosic papillae, providing structural defense in resistant tomato and alfalfa (Street et al., 1986; Newcombe against various pathogens (Schneider et al., 2016). In addition, and Robb, 1988; Robb et al., 1991). Similarly, induction of su- callose can constitute a matrix for accumulation of antimicro- berin deposition is an important line of defense in Ulmus minor bial compounds, thereby providing targeted delivery of chem- against O. novo-ulmi (Martín et al., 2008). Interestingly, ex- ical defenses at the sites of pathogen attack (Luna et al., 2011). ogenous application of phenolic compounds further increased In the vasculature, callose has also been shown to act as a resistance of trees to this pathogen through formation of structural barrier against fungal wilt pathogens, restricting suberin-like compounds in xylem tissues (Martín et al., 2008). their horizontal vessel-to-vessel movement. Tomato plants re- Another example of xylem tissue suberization induced by a sistant to F. oxysporum f. sp. lycopersici form callose deposits in vascular pathogen is provided by the histological characteriza- paravascular parenchyma cells and at pit membranes in response tion of grapevine infection by the wilt fungus Phaeomoniella to infection by this pathogen (Beckman et al., 1982; Mueller chlamydospora (Pouzoulet et al., 2013, 2017). It was shown that and Beckman, 1988). In addition, application of the microbe- deposition of suberin in paravascular parenchyma cells is an associated molecular pattern chitosan, a derivative of chitin, re- effective barrier against horizontal P. chlamydospora coloniza- stricts colonization of F. oxysporum f.sp. lycopersici by inducing a tion from one vessel to the adjacent vessel. Suberin was also vascular coating composed of callose and phenolic compounds shown to form deposits in tyloses induced by infection with (Benhamou et al., 1994). Furthermore, cotton roots infected this pathogen (Pouzoulet et al., 2013). Interestingly, inhibition with V. dahliae showed reinforcement with callose deposits of tylose formation in grapevine by P. chlamydospora led to vas- (Daayf et al., 1997). In contrast, infection by the bacterial wilt cular coating of surrounding parenchyma cells with phenolic pathogen R. solanacearum caused deposition of callose in both compounds, including suberin (Pouzoulet et al., 2017).This in- tolerant and susceptible potato plants, indicating that in this dicates that sequential/superimposed defense mechanisms are interaction callose may not be as important for resistance to- in place to restrict pathogen progression to the infection site, wards the pathogen (Ferreira et al., 2017). Additional research once it has reached the vasculature. is needed to clarify the precise role and regulation of callose Although suberin deposition seems to be an important com- as a structural defense mechanism induced upon perception of ponent of defense responses against vascular pathogens, very vascular wilt pathogens. 194 | Kashyap et al. Concluding remarks and future prospects Raman spectroscopy or MALDI (matrix-assisted laser desorp- tion ionization) spectrometry imaging, both of which can be During the last few decades, much evidence has accumulated extremely useful in zonal responses such as structural resistance. showing the importance of physico-chemical barriers as a cru- All this knowledge could lead in the future to the engineering cial component of resistance towards xylem vascular patho- of metabolic pathways of vascular coating compounds in spe- gens. From all the research until now in this field, it becomes cific cells, to produce resistant plants against xylem colonizers. clear that the localization and timing of formation of these With this article we hope to contribute towards raising vascular structures is key for their effectiveness as barriers for awareness of the importance of attaining a better understanding pathogen confinement. Resistant plants are able to form ver- of the structural physico-chemical barriers as a crucial compo- tical and horizontal barriers quickly upon pathogen invasion of nent of resistance towards xylem vascular pathogens. Disease the vasculature, confining them to infected vessels and avoiding management through host resistance is the most efficient and spread to the rest of the plant. In susceptible plants, formation eco-friendly approach to control pathogens. However, a lot of the same vascular structures is observed, but not targeted to has to be learnt about the complex genetic interactions which infected vessels, and later in time, once the pathogen has spread govern induced structural resistance in various hosts, to be able throughout the plant they have no effect on disease progression. to deploy this trait in future cultivars and fight vascular patho- However, the mechanisms regulating the spatial and temporal gens, agents of the most devastating plant diseases in the field. formation of vascular structures leading to effective pathogen confinement, and their precise composition, are old questions that remain unanswered. In fact, it remains unclear as to how Acknowledgements vascular wilt pathogens are perceived at the vasculature, and how this perception is transduced into timely and restricted The authors would like to thank all members of the Bacterial plant dis- formation of structural defenses. Since effective mechanisms of eases and cell death lab for helpful comments.We apologize to all authors whose work has been omitted because of space limitations. Research in the resistance are very much sought after in breeding programs, in lab is funded by the Spanish Ministry of Economy and Competitiveness the coming years it will be important to make an effort to ad- with grants 2016-78002-R (AGL) and RyC 2014–16158 (NSC), by the vance knowledge in this area, even though inducible structural Ministry of Science and Innovation / Spanish State Research Agency defenses are governed by complex polygenic traits. PID2019-108595RB-I00 / AEI / 10.13039/501100011033, and Major technological advances in the last few years have through the “Severo Ochoa Programme for Centres of Excellence in placed plant molecular biologists in a privileged position to R&D” (SEV-2015-0533). AK is the recipient of a Netaji Subhas—Indian make significant advances. Of particularly relevance is the Council of Agricultural Research (ICAR) International Fellowship. This CRISPR-Cas9 technology, which has proven extremely ef- work was also supported by the CERCA Programme / Generalitat de ficient for Solanaceous crops such as tomato, pepper and Catalunya. eggplant, which are severely affected by wilt diseases caused by xylem vascular pathogens (Hu et al., 2019; Li et al., 2019; Wang et al., 2019). Importantly, an array of technologies have Author contribution emerged that allow the study of specific processes in a cell, or AK, MP-M, MC, MV, and NSC conceptualized and wrote the article, in a tissue-specific manner. These techniques will become in- and obtained funds. strumental in the study of plant-pathogen interactions, which constitute a localized phenomenon by its very nature. This is particularly the case for colonization of vascular cells by vas- References cular pathogens, since the ability to confine the invading agent Alassimone J, Fujita S, Doblas VG, et al. 2016. Polarly localized kinase is a key feature of resistant plants. With the advent of single- SGN1 is required for Casparian strip integrity and positioning. Nature Plants cell technologies it will be possible to attain astonishing reso- 2, 16113. lution when investigating the processes occurring at infected Álvarez B, Biosca EG, López MM. 2010. On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. In: Méndez-Vilas A, cells and surrounding areas. For instance, RNA sequencing ed. Technology and education topics in applied microbiology and micro- of laser-dissected areas or single cells allows profiling of the bial biotechnology. Current Research, Technology and Education Topics in transcriptomic landscape after infection at relevant sites. In Applied Microbiology. Badajoz: Formatex, 267–279. turn, this will allow the identification of marker genes associ- Antanaviciute L, Šurbanovski N, Harrison N, McLeary KJ, Simpson DW, Wilson F, Sargent DJ, Harrison RJ. 2015. Mapping QTL ated with the formation of structural defenses and the subse- associated with Verticillium dahliae resistance in the cultivated strawberry quent generation of transgenic marker lines, to be able to track (Fragaria × ananassa). Horticulture Research 2, 15009. relevant cells/tissues at early time points after infection for their Araujo L, Bispo WM, Cacique IS, Moreira WR, Rodrigues FÁ. analysis. In addition, extremely sensitive analytical techniques 2014. Resistance in mango against infection by Ceratocystis fimbriata. Phytopathology 104, 820–833. have been developed in recent years that allow the identifica- Baayen RP, Elgersma DM. 1985. Colonization and histopathology of sus- tion and quantification of proteins, small molecules and me- ceptible and resistant carnation cultivars infected with Fusarium oxysporum tabolites, and the interactions between them. This includes f. sp. dianthi. Netherlands Journal of Plant Pathology 91, 119–135. Inducible barriers against plant vascular wilt pathogens | 195 Bae C, Han SW, Song YR, Kim BY, Lee HJ, Lee JM, Yeam I, Heu S, Choat B, Cobb AR, Jansen S. 2008. Structure and function of bordered Oh CS. 2015. Infection processes of xylem-colonizing pathogenic bacteria: pits: new discoveries and impacts on whole-plant hydraulic function. New possible explanations for the scarcity of qualitative disease resistance genes Phytologist 177, 608–625. against them in crops. Theoretical and Applied Genetics 128, 1219–1229. Clérivet A, Déon V, Alami I, Lopez F, Geiger JP, Nicole M. 2000. Bani M, Pérez-De-Luque A, Rubiales D, Rispail N. 2018. Physical and Tyloses and gels associated with cellulose accumulation in vessels are re- chemical barriers in root tissues contribute to quantitative resistance to sponses of plane tree seedlings (Platanus x acerifolia) to the vascular fungus Fusarium oxysporum f. sp. pisi in pea. Frontiers in Plant Science 9, 199. Ceratocystis fimbriata f. sp platani. Trees 15, 25–31. Barberon M, Vermeer JEM, De Bellis D, Wang P, Naseer S, Cochard H, Tyree MT. 1990. Xylem dysfunction in Quercus: vessel sizes, Andeersen TG, Humbel BM, Nawrath C, Takano J, Salt DE. 2016. tyloses, cavitation and seasonal changes in embolism. Tree Physiology 6, Adaptation of root function by nutrient-induced plasticity of endodermal dif- 393–407. ferentiation. Cell 164, 447–459. Cohen H, Fedyuk V, Wang C, Wu S, Aharoni A. 2020. SUBERMAN regu- Beckman CH, Mueller WC, Tessier BJ, Harrison NA. 1982. Recognition lates developmental suberization of the Arabidopsis root endodermis. The and callose deposition in response to vascular infection in Fusarium wilt- Plant Journal 102, 431–447. resistant or susceptible tomato plants. Physiological Plant Pathology 20, Cooper RM, Williams JS. 2004. Elemental sulphur as an induced 1–10. antifungal substance in plant defence. Journal of Experimental Botany 55, Beckman CH, Roberts EM. 1995. On the nature and genetic basis for 1947–1953. resistance and tolerance to fungal wilt diseases of plants. Advances in Cottle W, Kolattukudy PE. 1982. Abscisic acid stimulation of suberiza- Botanical Research 21, 35–77. tion: induction of enzymes and deposition of polymeric components and Beisson F, Li Y, Bonaventure G, Pollard M, Ohlrogge JB. 2007. The associated waxes in tissue cultures of potato tuber. Plant Physiology 70, acyltransferase GPAT5 is required for the synthesis of suberin in seed coat 775–780. and root of Arabidopsis. The Plant cell 19, 351–368. Daayf F, Nicole M, Boher B, Pando A, Geiger JP. 1997. Early vascular Beisson F, Li-Beisson Y, Pollard M. 2012. Solving the puzzles of cutin defense reactions of cotton roots infected with a defoliating mutant strain and suberin polymer biosynthesis. Current Opinion in Plant Biology 15, of Verticillium dahliae. European Journal of Plant Pathology 103, 125–136. 329–337. Davison EM, Tay FCS. 1985. The effect of waterlogging on seedlings of Benhamou N, Lafontaine P, Nicole M. 1994. Induction of systemic re- Eucalyptus marginata. New Phytologist 101, 743–753. sistance to Fusarium crown and root rot in tomato plants by seed treatment De Ascensao AR, Dubery IA. 2000. Panama disease: cell wall reinforce- with chitosan. Phytopathology 84, 1432–1444. ment in banana roots in response to elicitors from Fusarium oxysporum Benhamou N. 1995. Ultrastructural and cytochemical aspects of the re- f. sp. cubense race four. Phytopathology 90, 1173–1180. sponse of eggplant parenchyma cells in direct contact with Verticillium- de Jonge R, van Esse HP, Maruthachalam K, et al. 2012. Tomato im- infected xylem vessels. Physiological and Molecular Plant Pathology 46, mune receptor Ve1 recognizes effector of multiple fungal pathogens un- 321–338. covered by genome and RNA sequencing. Proceedings of the National Bernards MA. 2002. Demystifying suberin. Canadian Journal of Botany Academy of Sciences, USA 109, 5110–5115. 80, 227–240. De Micco V, Balzano A, Wheeler EA, Baas P. 2016. Tyloses and gums: Bernards MA. 2018. Differential induction of polar and non-polar metab- a review of structure, function and occurrence of vessel occlusions. IAWA olism during wound-induced suberization in potato (Solanum tuberosum L.) Journal 37, 186–205. tubers. The Plant Journal 93, 931–942. Deslandes L, Olivier J, Theulieres F, Hirsch J, Feng DX, Bittner- Bishop CD, Cooper RM. 1983. An ultrastructural study of root invasion in Eddy P, Beynon J, Marco Y. 2002. Resistance to Ralstonia solanacearum three vascular wilt diseases. Physiological Plant Pathology 22, 15–27. in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a Bishop CD, Cooper RM. 1984. Ultrastructure of vascular colonization by member of a novel family of resistance genes. Proceedings of the National fungal wilt pathogens. II. Invasion of resistant cultivars. Physiological Plant Academy of Sciences, USA 99, 2404–2409. Pathology 24, 277–289. Deyett E, Pouzoulet J, Yang JI, Ashworth VE, Castro C, Roper MC, Blaedow RA, Juzwik J. 2010. Spatial and temporal distribution of Rolshausen PE. 2019. Assessment of Pierce’s disease susceptibility in Vitis Ceratocystis fagacearum in roots and root grafts of oak wilt affected red vinifera cultivars with different pedigrees. Plant Pathology 68, 1079–1087. oaks. Arboriculture and Urban Forestry 36, 28–34. Diener AC, Ausubel FM. 2005. RESISTANCE TO FUSARIUM Bonsen KJM, Kucera LJ. 1990. Vessel occlusions in plants: morpho- OXYSPORUM 1, a dominant Arabidopsis disease-resistance gene, is not logical, functional and evolutionary aspects. IAWA Bulletin 11, 393–399. race specific. Genetics 171, 305–321. Bu B, Qiu D, Zeng H, Guo L, Yuan J, Yang X. 2014. A fungal pro- Dixon RA, Paiva NL. 1995. Stress-induced phenylpropanoid metabolism. tein elicitor PevD1 induces Verticillium wilt resistance in cotton. Plant Cell The Plant Cell 7, 1085–1097. Reports 33, 461–470. Doblas VG, Smakowska-Luzan E, Fujita S, Alassimone J, Barberon M, Bubán T, Orosz-Kovács Z, Farkas Á. 2003. The nectary as the primary Madalinski M, Belkhadir Y, Geldner N. 2017. Root diffusion barrier con- site of infection by Erwinia amylovora (Burr.) Winslow et al.: A mini review. trol by a vasculature-derived peptide binding to the SGN3 receptor. Science Plant Systematics and Evolution 238, 183–194. 355, 280–284. Carmeille A, Caranta C, Dintinger J, Prior P, Luisetti J, Besse P. 2006. Ellinger D, Voigt CA. 2014. Callose biosynthesis in Arabidopsis with a Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resist- focus on pathogen response: what we have learned within the last decade. ance in tomato. Theoretical and Applied Genetics 113, 110–121. Annals of Botany 114, 1349–1358. Catanzariti AM, Do HT, Bru P, de Sain M, Thatcher LF, Rep M, Eynck C, Koopmann B, Karlovsky P, von Tiedemann A. 2009. Internal Jones DA. 2017. The tomato I gene for Fusarium wilt resistance encodes resistance in winter oilseed rape inhibits systemic spread of the vascular an atypical leucine-rich repeat receptor-like protein whose function is never- pathogen Verticillium longisporum. Phytopathology 99, 802–811. theless dependent on SOBIR1 and SERK3/BAK1. The Plant Journal 89, Ferreira V, Pianzzola MJ, Vilaró FL, Galván GA, Tondo ML, 1195–1209. Rodriguez MV, Orellano EG, Valls M, Siri MI. 2017. Interspecific potato Catanzariti AM, Lim GT, Jones DA. 2015. The tomato I-3 gene: a novel breeding lines display differential colonization patterns and induced defense gene for resistance to Fusarium wilt disease. New Phytologist 207, 106–118. responses after Ralstonia solanacearum infection. Frontiers in Plant Science Chen YC, Wong CL, Muzzi F, Vlaardingerbroek I, Kidd BN, Schenk PM. 8, 1424. 2014. Root defense analysis against Fusarium oxysporum reveals new Franke R, Höfer R, Briesen I, Emsermann M, Efremova N, regulators to confer resistance. Scientific Reports 4, 5584. Yephremov A, Schreiber L. 2009. The DAISY gene from Arabidopsis 196 | Kashyap et al. encodes a fatty acid elongase condensing enzyme involved in the bio- Lambel S, Lanini B, Vivoda E, Fauve J, Patrick Wechter W, Harris- synthesis of aliphatic suberin in roots and the chalaza-micropyle region of Shultz KR, Massey L, Levi A. 2014. A major QTL associated with seeds. The Plant Journal 57, 80–95. Fusarium oxysporum race 1 resistance identified in genetic populations Fujita S, De Bellis D, Edel KH, et al. 2020. SCHENGEN receptor module derived from closely related watermelon lines using selective genotyping drives localized ROS production and lignification in plant roots. The EMBO and genotyping-by-sequencing for SNP discovery. Theoretical and Applied Journal 39, e103894. Genetics 127, 2105–2115. Galindo-González L, Deyholos MK. 2016. RNA-seq transcriptome re- Lashbrooke J, Cohen H, Levy-Samocha D, et al. 2016. MYB107 and sponse of flax (Linum usitatissimum L.) to the pathogenic fungus Fusarium MYB9 homologs regulate suberin deposition in angiosperms. The Plant Cell oxysporum f. sp. lini. Frontiers in Plant Science 7, 1766. 28, 2097–2116. Gou M, Hou G, Yang H, Zhang X, Cai Y, Kai G, Liu CJ. 2017. The Le Roux C, Huet G, Jauneau A, et al. 2015. A receptor pair with an in- MYB107 transcription factor positively regulates suberin biosynthesis. Plant tegrated decoy converts pathogen disabling of transcription factors to im- Physiology 173, 1045–1058. munity. Cell 161, 1074–1088. Gramaje D, Úrbez-Torres JR, Sosnowski MR. 2018. Managing grape- Lee MH, Jeon HS, Kim SH, Chung JH, Roppolo D, Lee HJ, Cho HJ, vine trunk diseases with respect to etiology and epidemiology: current strat- Tobimatsu Y, Ralph J, Park OK. 2019. Lignin-based barrier restricts egies and future prospects. Plant Disease 102, 12–39. pathogens to the infection site and confers resistance in plants. The EMBO Journal 38, e101948. Grimault V, Gélie B, Lemattre M, Prior P, Schmit J. 1994. Comparative histology of resistant and susceptible tomato cultivars infected by Leśniewska J, Öhman D, Krzesłowska M, Kushwah S, Barciszewska- Pseudomonas solanacearum. Physiological and Molecular Plant Pathology Pacak M, Kleczkowski LA, Sundberg B, Moritz T, Mellerowicz EJ. 44, 105–123. 2017. Defense responses in aspen with altered pectin methylesterase activity reveal the hormonal inducers of tyloses. Plant Physiology 173, Gui YJ, Chen JY, Zhang DD, et al. 2017. Verticillium dahliae manipu- 1409–1419. lates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1. Environmental Microbiology 19, Li R, Liu C, Zhao R, Wang L, Chen L, Yu W, Zhang S, Sheng J, Shen L. 1914–1932. 2019. CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance. BMC Plant Biology 19, 1–13. Höfer R, Briesen I, Beck M, Pinot F, Schreiber L, Franke R. 2008. The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J. 2011. omega-hydroxylase involved in suberin monomer biosynthesis. Journal of Callose deposition: a multifaceted plant defense response. Molecular Plant- Experimental Botany 59, 2347–2360. Microbe Interactions 24, 183–193. Houterman PM, Ma L, van Ooijen G, de Vroomen MJ, Cornelissen BJ, Mangin B, Thoquet P, Olivier J, Grimsley NH. 1999. Temporal and mul- Takken FL, Rep M. 2009. The effector protein Avr2 of the xylem-colonizing tiple quantitative trait loci analyses of resistance to bacterial wilt in tomato fungus Fusarium oxysporum activates the tomato resistance protein I-2 permit the resolution of linked loci. Genetics 151, 1165–1172. intracellularly. The Plant Journal 58, 970–978. Martín JA, Solla A, Coimbra MA, Gil L. 2005. Metabolic distinction of Hu N, Xian Z, Li N, Liu Y, Huang W, Yan F, Su D, Chen J, Li Z. 2019. Ulmus minor xylem tissues after inoculation with Ophiostoma novo-ulmi. Rapid and user-friendly open-source CRISPR/Cas9 system for single- or Phytochemistry 66, 2458–2467. multi-site editing of tomato genome. Horticulture Research 6, 7. Martín JA, Solla A, Domingues MR, Coimbra MA, Gil L. 2008. Hutson RA, Smith IM. 1980. Phytoalexins and tyloses in tomato culti- Exogenous phenol increase resistance of Ulmus minor to Dutch elm dis- vars infected with Fusarium oxysporum f.sp. lycopersici or Verticillium albo- ease through formation of suberin-like compounds on xylem tissues. atrum. Physiological Plant Pathology 17, 245–257. Environmental and Experimental Botany 64, 97–104. Ishihara T, Mitsuhara I, Hideki T, Kazuhiro N. 2012. Transcriptome Martin JA, Solla A, Woodward S, Gil L. 2007. Detection of differen- analysis of quantitative resistance- specific response upon Ralstonia tial changes in lignin composition of elm xylem tissues inoculated with solanacearum infection in tomato. PLoS Pathogens 7, e46763. Ophiostoma novo-ulmi using fourier transform-infrared spectroscopy. Forest Pathology 37, 187–191. Jones JD, Dangl JL. 2006. The plant immune system. Nature 444, 323–329. Mes JJ, van Doorn AA, Wijbrandi J, Simons G, Cornelissen BJ, Haring MA. 2000. Expression of the Fusarium resistance gene I-2 Joobeur T, King JJ, Nolin SJ, Thomas CE, Dean RA. 2004. The colocalizes with the site of fungal containment. The Plant Journal 23, Fusarium wilt resistance locus Fom-2 of melon contains a single resistance 183–193. gene with complex features. The Plant Journal 39, 283–297. Milling A, Babujee L, Allen C. 2011. Ralstonia solanacearum extracellular Kang X, Kirui A, Dickwella Widanage MC, Mentink-Vigier F, polysaccharide is a specific elicitor of defense responses in wilt-resistant Cosgrove DJ, Wang T. 2019. Lignin-polysaccharide interactions in plant tomato plants. PLoS One 6, e15853. secondary cell walls revealed by solid-state NMR. Nature Communications 10, 347. Morello P, Díez CM, Codes M, Rallo L, Barranco D, Trapero A, Moral J. 2016. Sanitation of olive plants infected by Verticillium dahliae Katagiri F, Tsuda K. 2010. Understanding the plant immune system. using heat treatments. Plant Pathology 65, 412–421. Molecular Plant-Microbe Interactions 23, 1531–1536. Mueller W, Beckman C. 1988. Correlated light and electron microscope Kim SG, Hur OS, Ro NY, Ko HC, Rhee JH, Sung JS, Ryu KY, Lee SY, studies of callose deposits in vascular paerenchyma cells of tomato plants Baek HJ. 2016. Evaluation of resistance to Ralstonia solanacearum in to- inoculated with Fusarium oxysporum f.sp. lycopersici. Physiological and mato genetic resources at seedling stage. The Plant Pathology Journal 32, Molecular Plant Pathology 33, 201–208. 58–64. Nakaho K, Hibino H, Miyagawa H. 2000. Possible mechanisms limiting Kosma DK, Murmu J, Razeq FM, Santos P, Bourgault R, Molina I, movement of Ralstonia solanacearum in resistant tomato tissues. Journal of Rowland O. 2014. AtMYB41 activates ectopic suberin synthesis and Phytopathology 148, 181–190. assembly in multiple plant species and cell types. The Plant Journal 80, Negrel J, Lotfy S, Javelle F. 1995. Modulation of the activity of two 216–229. hydroxycinnamoyl transferases in wound-healing potato tuber discs in Kpemoua K, Boher B, Nicole M, Calatayud P, Geiger J. 1996. response to pectinase or abscisic acid. Journal of Plant Physiology 146, Cytochemistry of defense responses in cassava infected by Xanthomonas 318–322. campestris pv. manihotis. Canadian Journal of Microbiology 42, 1131–1143. Newcombe G, Robb J. 1988. The function and relative importance of Krivanek AF, Riaz S, Walker MA. 2006. Identification and molecular the vascular coating response in highly resistant, moderately resistant and mapping of PdR1 a primary resistance gene to Pierce’s disease in Vitis. susceptible alfalfa infected by Verticillium albo-atrum. Physiological and Theoretical and Applied Genetics 112, 1125–1131. Molecular Plant Pathology 33, 47–58. Inducible barriers against plant vascular wilt pathogens | 197 Ngou BPM, Ahn H, Ding P, Jones JDG. 2020. Mutual potentiation of Renzi M, Copini P, Taddei AR, Rossetti A, Gallipoli L, Mazzaglia A, plant immunity by cell-surface and intracellular receptors. BioRxiv doi. Balestra GM. 2012. Bacterial canker on kiwifruit in Italy: anatomical org/10.1101/2020.04.10.034173 1–29. [Preprint]. changes in the wood and in the primary infection sites. Phytopathology 102, Nicholson RL. 1992. Phenolic compounds and their role in disease resist- 827–840. ance. Annual Reviews in Phytopathology 30, 369–389. Rioux D, Blais M, Nadeau-Thibodeau N, Lagacé M, DesRochers P, Niemann AH. 1994. A crucial role of phenolic metabolism in resistance of Klimaszewska K, Bernier L. 2018. First extensive microscopic study carnations to wilt diseases. Acta Horticulturae 381, 565–571. of butternut defense mechanisms following inoculation with the canker pathogen Ophiognomonia clavigignenti-juglandacearum reveals compart- Nishimura MT, Dangl JL. 2010. Arabidopsis and the plant immune mentalization of tissue damage. Phytopathology 108, 1237–1252. system. The Plant Journal 61, 1053–1066. Rioux D, Nicole M, Simard M, Ouellette GB. 1998. Immunocytochemical Novo M, Silvar C, Merino F, Martínez-Cortés T, Lu F, Ralph J, Pomar F. evidence that secretion of pectin occurs during gel (Gum) and tylosis forma- 2017. Deciphering the role of the phenylpropanoid metabolism in the toler- tion in trees. Phytopathology 88, 494–505. ance of Capsicum annuum L. to Verticillium dahliae Kleb. Plant Science 258, 12–20. Robb J, Lee B, Nazar RN. 2007. Gene suppression in a tolerant tomato- vascular pathogen interaction. Planta 226, 299–309. Palanga KK, Jamshed M, Rashid MHO, et al. 2017. Quantitative Trait Locus mapping for Verticillium wilt resistance in an upland cotton recom- Robb J, Lee SW, Mohan R, Kolattukudy PE. 1991. Chemical character- binant inbred line using SNP-based high density genetic map. Frontiers in ization of stress-induced vascular coating in tomato. Plant Physiology 97, Plant Science 8, 382. 528–536. Park CJ, Ronald PC. 2012. Cleavage and nuclear localization of the rice Sabbavarapu MM, Sharma M, Chamarthi SK, et al. 2013. Molecular XA21 immune receptor. Nature Communications 3, 920. mapping of QTLs for resistance to Fusarium wilt (race 1) and Ascochyta Pastrana AM, Watson DC, Gordon TR. 2019. Transmission of Fusarium blight in chickpea (Cicer arietinum L.). Euphytica 193, 121–133. oxysporum f. sp. fragariae through stolons in strawberry plants. Plant Sabella E, Luvisi A, Aprile A, Negro C, Vergine M, Nicolì F, Miceli A, Disease 103, 1249–1251. De Bellis L. 2018. Xylella fastidiosa induces differential expression of lignifi- Planas-Marquès M, Kressin JP, Kashyap A, Panthee DR, Louws FJ, cation related-genes and lignin accumulation in tolerant olive trees ‘Leccino’. Coll NS, Valls M. 2019. Four bottlenecks restrict colonization and inva- Journal of Plant Physiology 220, 60–68. sion by the pathogen Ralstonia solanacearum in resistant tomato. Journal Sanderlin RS, Melanson RA. 2006. Transmission of Xylella fastidiosa of Experimental Botany 71, 2157–2171. through pecan rootstock. HortScience 41, 1455–1456. Plichta R, Urban J, Gebauer R, Dvořák M, Ďurkovič J. 2016. Long-term Sarris PF, Duxbury Z, Huh SU, et al. 2015. A plant immune receptor de- impact of Ophiostoma novo-ulmi on leaf traits and transpiration of branches tects pathogen effectors that target WRKY transcription factors. Cell 161, in the Dutch elm hybrid ‘Dodoens’. Tree Physiology 36, 335–344. 1089–1100. Pouzoulet J, Jacques A, Besson X, Dayde J, Mailhac N. 2013. Schneider R, Hanak T, Persson S, Voigt CA. 2016. Cellulose and callose Histopathological study of response of Vitis vinifera ‘Cabernet Sauvignon’ synthesis and organization in focus, what’s new? Current Opinion in Plant to bark and wood injury with and without inoculation by Phaeomoniella Biology 34, 9–16. chlamydospora. Phytopathologia Mediterranea 52, 313–323. Schornack S, Ballvora A, Gürlebeck D, Peart J, Baulcombe D, Pouzoulet J, Pivovaroff AL, Santiago LS, Rolshausen PE. 2014. Can Ganal M, Baker B, Bonas U, Lahaye T. 2004. The tomato resistance vessel dimension explain tolerance toward fungal vascular wilt diseases in protein Bs4 is a predicted non-nuclear TIR-NB-LRR protein that medi- woody plants? Lessons from Dutch elm disease and esca disease in grape- ates defense responses to severely truncated derivatives of AvrBs4 and vine. Frontiers in Plant Science 5, 253. overexpressed AvrBs3. The Plant Journal: for cell and molecular biology Pouzoulet J, Rolshausen P, Charbois R, Chen J, Guillaumie S, Ollat N, 37, 46–60. Gambetta GA, Delmas CEL. 2020. Behind the curtain of the compartmen- talization process: exploring how xylem vessel diameter impacts vascular Seo Y, Kim YH. 2017. Pathological interrelations of soil-borne diseases pathogen resistance. Plant, Cell & Environment, doi: 10.1111/pce.13848. in Cucurbits caused by Fusarium species and Meloidogyne incognita. The Plant Pathology Journal 33, 410–423. Pouzoulet J, Scudiero E, Schiavon M, Rolshausen PE. 2017. Xylem vessel diameter affects the compartmentalization of the vascular pathogen Serra O, Hohn C, Franke R, Prat S, Molinas M, Figueras M. 2010. Phaeomoniella chlamydospora in grapevine. Frontiers in Plant Science 8, A feruloyl transferase involved in the biosynthesis of suberin and suberin- 1442. associated wax is required for maturation and sealing properties of potato periderm. The Plant journal 62, 277–290. Pouzoulet J, Scudiero E, Schiavon M, Santiago LS, Rolshausen PE. 2019. Modeling of xylem vessel occlusion in grapevine. Tree Physiology 39, Shi J, Mueller WC, Beckman CH. 1991. Ultrastructural responses of 1438–1445. vessel contact cells in cotton plants resistant or susceptible to infection by Fusarium oxysporum f. sp. vasinfectum. Physiological and Molecular Plant Pradhan BB, Ranjan M, Chatterjee S. 2012. XadM, a novel adhesin of Pathology 38, 211–222. Xanthomonas oryzae pv. oryzae, exhibits similarity to Rhs family proteins and is required for optimum attachment, biofilm formation, and virulence. Shigo AL, Marx HG. 1977. Compartmentalization of decay in trees. U. S. Molecular Plant-Microbe Interactions 25, 1157–1170. Department of Agriculture. Agriculture Information Bulletin. 405, 1–73. Prakasha A, Darren Grice I, Vinay Kumar KS, Sadashiva MP, Soliday CL, Dean BB, Kolattukudy PE. 1978. Suberization: inhibition by Shankar HN, Umesha S. 2017. Extracellular polysaccharide from Ralstonia washing and stimulation by abscisic acid in potato disks and tissue culture. solanacearum; a strong inducer of eggplant defense against bacterial wilt. Plant Physiology 61, 170–174. Biological Control 110, 107–116. Song Y, Liu L, Wang Y, Valkenburg DJ, Zhang X, Zhu L, Thomma BPHJ. Ranathunge K, Schreiber L, Franke R. 2011. Suberin research in the 2018. Transfer of tomato immune receptor Ve1 confers Ave1-dependent genomics era–new interest for an old polymer. Plant Science 180, 399–413. Verticillium resistance in tobacco and cotton. Plant Biotechnology Journal 16, 638–648. Rapicavoli JN, Blanco-Ulate B, Muszyński A, Figueroa-Balderas R, Morales-Cruz A, Azadi P, Dobruchowska JM, Castro C, Cantu D, Stone BA. 2009. Chemistry of β-glucans. In: Bacic A, Fincher G, Stone B, Roper MC. 2018. Lipopolysaccharide O-antigen delays plant innate im- eds. Chemistry, biochemistry and biology of 1–3 Beta glucans and related mune recognition of Xylella fastidiosa. Nature Communications 9, 390. polysaccharides. San Diego, USA: Academic press, 5–46. Ratnayake K, Joyce DC, Webb RI. 2013. Cu2+ inhibition of gel secretion Street PFS, Robb J, Ellis BE. 1986. Secretion of vascular coating com- in the xylem and its potential implications for water uptake of cut Acacia ponents by xylem parenchyma cells of tomatoes infected with Verticillium holosericea stems. Physiologia Plantarum 148, 538–548. albo-atrum. Protoplasma 132, 1–11. 198 | Kashyap et al. Sun Q, Greve LC, Labavitch JM. 2011. Polysaccharide compositions resistance of tomato cultivar ‘Hawaii 7996’ to Ralstonia solanacearum. of intervessel pit membranes contribute to Pierce’s disease resistance of Euphytica 190, 241–252. grapevines. Plant Physiology 155, 1976–1987. Wang JF, Olivier J, Thoquet P, Mangin B, Sauviac L, Grimsley NH. Sun Q, Rost TL, Matthews MA. 2008. Wound-induced vascular oc- 2000. Resistance of tomato line Hawaii7996 to Ralstonia solanacearum clusions in Vitis vinifera (Vitaceae): tyloses in summer and gels in winter. Pss4 in Taiwan is controlled mainly by a major strain-specific locus. American Journal of Botany 95, 1498–1505. Molecular Plant-Microbe Interactions 13, 6–13. Sun Q, Rost TL, Reid MS, Matthews MA. 2007. Ethylene and not em- Wang L, Albert M, Einig E, Fürst U, Krust D, Felix G. 2016. The pattern- bolism is required for wound-induced tylose development in stems of recognition receptor CORE of Solanaceae detects bacterial cold-shock pro- grapevines. Plant Physiology 145, 1629–1636. tein. Nature Plants 2, 16185. Sun Q, Sun Y, Walker MA, Labavitch JM. 2013. Vascular occlusions Wang P, Lee Y, Igo MM, Roper MC. 2017. Tolerance to oxidative stress in grapevines with Pierce’s disease make disease symptom development is required for maximal xylem colonization by the xylem-limited bac- worse. Plant Physiology 161, 1529–1541. terial phytopathogen, Xylella fastidiosa. Molecular Plant Pathology 18, Thomma BPHJ, Nürnberger T, Joosten MHAJ. 2011. Of PAMPs and 990–1000. effectors: The blurred PTI-ETI dichotomy. Plant Cell 23, 4–15. Wang T, Zhang H, Zhu H. 2019. CRISPR technology is revolutionizing the Thoquet P, Olivier J, Sperisen C, Rogowsky P, Laterrot H, Grimsley N. improvement of tomato and other fruit crops. Horticulture Research 6, 77. 1996. Quantitative trait loci determining resistance to bacterial wilt in tomato Wei Y, Balaceanu A, Rufian JS, Segonzac C, Zhao A, Morcillo RJL, ‘Hawaii7996’. Molecular Plant-Microbe Interactions 9, 826–836. Macho AP. 2020. An immune receptor complex evolved in soybean to per- Tjamos EC, Smith IM. 1975. The expression of resistance to Verticillium ceive a polymorphic bacterial flagellin. Nature Communications 11, 3763. albo-atrum in monogenically resistant tomato varieties. Physiological Plant Wei Y, Caceres-Moreno C, Jimenez-Gongora T, Wang K, Sang Y, Pathology 6, 215–225. Lozano-Duran R, Macho AP. 2018. The Ralstonia solanacearum csp22 Triplett LR, Cohen SP, Heffelfinger C, Schmidt CL, Huerta AI, peptide, but not flagellin-derived peptides, is perceived by plants from the Tekete C, Verdier V, Bogdanove AJ, Leach JE. 2016. A resistance locus Solanaceae family. Plant Biotechnology Journal 16, 1349–1362. in the American heirloom rice variety Carolina Gold Select is triggered by Williams JS, Hall SA, Hawkesford MJ, Beale MH, Cooper RM. 2002. TAL effectors with diverse predicted targets and is effective against African Elemental sulfur and thiol accumulation in tomato and defense against a strains of Xanthomonas oryzae pv. oryzicola. The Plant Journal 87, 472–483. fungal vascular pathogen. Plant Physiology 128, 150–159. Ulloa M, Hutmacher RB, Roberts PA, Wright SD, Nichols RL, Woolfson KN, Haggitt ML, Zhang Y, et al. 2011. Lignin metabolism has Michael Davis R. 2013. Inheritance and QTL mapping of Fusarium wilt race a central role in the resistance of cotton to the wilt fungus Verticillium dahliae 4 resistance in cotton. Theoretical and Applied Genetics 126, 1405–1418. as revealed by RNA-seq-dependent transcriptional analysis and histochem- van der Does HC, Constantin ME, Houterman PM, Takken FLW, istry. Journal of Experimental Botany 62, 5607–5621. Cornelissen BJC, Haring MA, van den Burg HA, Rep M. 2019. Fusarium Woolfson KN. 2018. Suberin biosynthesis and deposition in the wound- oxysporum colonizes the stem of resistant tomato plants, the extent varying healing potato (Solanum tuberosum L.) tuber model. PhD thesis, The with the R-gene present. European Journal of Plant Pathology 154, 55–65. University of Western Ontario, London, Canada. https://ir.lib.uwo.ca/cgi/ Vander Molen GE, Beckman CH, Rodehorst E. 1987. The ultrastructure viewcontent.cgi?article=8075&context=etd. Accessed July 2020. of tylose formation in resistant banana following inoculation with Fusarium Xu L, Zhu L, Tu L, Liu L, Yuan D, Jin L, Long L, Zhang X. 2011. oxysporum f.sp. cubense. Physiological and Molecular Plant Pathology 31, Lignin metabolism has a central role in the resistance of cotton to the 185–200. wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent tran- Vander Molen GE, Beckman CH, Rodehorst E. 1977. Vascular gelation: scriptional analysis and histochemistry. Journal of Experimental Botany a general response phenomenon following infection. Physiological Plant 62, 5607–5621. Pathology 11, 95–100. Yadav V, Molina I, Ranathunge K, Castillo IQ, Rothstein SJ, Reed JW. Vasse J, Frey P, Trigalet A. 1995. Microscopic studies of intercel- 2014. ABCG transporters are required for suberin and pollen wall extracel- lular infection and protoxylem invasion of tomato roots by Pseudomonas lular barriers in Arabidopsis. The Plant Cell 26, 3569–3588. solanacearum. Molecular Plant-Microbe Interactions 8, 241–251. Yadeta KA, J Thomma BP. 2013. The xylem as battleground for plant Venturas M, López R, Martín JA, Gascó A, Gil L. 2014. Heritability of hosts and vascular wilt pathogens. Frontiers in Plant Science 4, 97. Ulmus minor resistance to Dutch elm disease and its relationship to vessel Yuan M, Jiang Z, Bi G, Nomura K, Liu M, He SY, Zhou J-M, Xin X-F. size, but not to xylem vulnerability to drought. Plant Pathology 63, 500–509. 2020. Pattern-recognition receptors are required for NLR-mediated plant Vishwanath SJ, Kosma DK, Pulsifer IP, Scandola S, Pascal S, immunity. BioRxiv doi.org/10.1101/2020.04.10.031294. [Preprint]. Joubès J, Dittrich-Domergue F, Lessire R, Rowland O, Domergue F. Zaini PA, Nascimento R, Gouran H, Cantu D, Chakraborty S, Phu M, 2013. Suberin-associated fatty alcohols in Arabidopsis: distributions in Goulart LR, Dandekar AM. 2018. Molecular profiling of Pierce’s disease roots and contributions to seed coat barrier properties. Plant Physiology outlines the response circuitry of Vitis vinifera to Xylella fastidiosa infection. 163, 1118–1132. Frontiers in Plant Science 9, 771. Wang C, Ulloa M, Duong T, Roberts PA. 2018. Quantitative Trait Loci Zhou F, Emonet A, Dénervaud Tendon V, Marhavy P, Wu D, Lahaye T, mapping of multiple independent loci for resistance to Fusarium oxysporum Geldner N. 2020. Co-incidence of damage and microbial patterns controls f. sp. vasinfectum Races 1 and 4 in an interspecific cotton population. localized immune responses in roots. Cell 180, 440–453.e18. Phytopathology 108, 759–767. Zhu W, Gao E, Shaban M, Wang Y, Wang H, Nie X, Zhu L. 2018. Wang G, Roux B, Feng F, et al. 2015. The decoy substrate of a pathogen GhUMC1, a blue copper-binding protein, regulates lignin synthesis effector and a pseudokinase specify pathogen-induced modified-self rec- and cotton immune response. Biochemical and Biophysical Research ognition and immunity in plants. Cell Host & Microbe 18, 285–295. Communications 504, 75–81. Wang JF, Ho FI, Truong HTH, Huang SM, Balatero CH, Dittapongpitch V, Zimmermann MH. 1979. The discovery of tylose formation by a Viennese Hidayati N. 2013. Identification of major QTLs associated with stable lady in 1845. IAWA BULLETIN, 2–3, 51-56.