Journal of Experimental Botany, Vol. 71, No. 6 pp. 2157–2171, 2020 doi:10.1093/jxb/erz562 Advance Access Publication December 24, 2019 RESEARCH PAPER Four bottlenecks restrict colonization and invasion by the pathogen Ralstonia solanacearum in resistant tomato Marc Planas-Marquès1,‡, , Jonathan P. Kressin2,3,†,‡, Anurag Kashyap1, Dilip R. Panthee3, Frank J. Louws2,4, Nuria S. Coll1,§,*, , and Marc Valls1,5,§,*, Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 1 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, 08193 Barcelona, Spain 2 Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA 3 Department of Horticultural Science, North Carolina State University, Mountain Horticultural Crops Research and Extension Center, Mills River, NC 28759, USA 4 Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA 5 Department of Genetics, University of Barcelona, 08028 Barcelona, Spain † Current address: Department of Breeding, Hortigenetics Research (S.E.Asia) Ltd, East-West Seed Co., Chiang Mai 50290, Thailand ‡ MPM and JPK should be considered joint first authors. § NSC and MV should be considered joint senior authors. * Correspondence: nuria.sanchezcoll@cragenomica.es or marcvalls@ub.edu Received 4 September 2019; Editorial decision 11 December 2019; Accepted 20 December 2019 Editor: Peter Bozhkov, Swedish University of Agricultural Sciences, Sweden ABSTRACT Ralstonia solanacearum is a bacterial vascular pathogen causing devastating bacterial wilt. In the field, resistance against this pathogen is quantitative and is available for breeders only in tomato and eggplant. To understand the basis of resistance to R. solanacearum in tomato, we investigated the spatio-temporal dynamics of bacterial col- onization using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We found four ‘bottlenecks’ that limit the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion, and radial apoplastic spread in the cortex. Radial invasion of cor- tical extracellular spaces occurred mostly at late disease stages but was observed throughout plant infection. This study shows that resistance is expressed in both root and shoot tissues, and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads out of the xylem, occupying the plant apoplast niche. Our work will help elucidate the com- plex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new precision tools to fight bacterial wilt in the field. Keywords: Bacterial wilt, disease resistance, Ralstonia solanacearum, tomato, vascular pathogen, xylem. Introduction Bacterial wilt caused by the Ralstonia solanacearum species com- through wounds, colonizes the xylem tissue, moves up into the plex is a disease of major economic importance, impacting the stem, and causes a rapid, permanent wilt through a combin- production of solanaceous crops, legumes, banana, ginger, and ation of high bacterial density and mass production of extra- ornamentals (Hayward, 1994). R. solanacearum enters the roots cellular polysaccharides (Hayward, 1991; Grimault and Prior, © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com 2158 | Planas-Marquès et al. 1993; McGarvey et al., 1999; Schell, 2000). R. solanacearum can both the primary and secondary xylem tissues of the suscep- move across the root following either an apoplastic pathway tible line Ponderosa (Nakaho, 1997a). Thus, disease severity in through the middle lamella or a pseudo-symplastic pathway via R. solanacearum-infected tomato plants was proposed to cor- the xylem vessel lumens and axillary pits (Schell, 2000). relate with the extent of bacterial invasion into the secondary The management of bacterial wilt is challenging due to the xylem tissues (Nakaho, 1997a, b). This limitation of pathogen aggressiveness of R. solanacearum and its broad geographical movement from the protoxylem or the primary xylem to other distribution, wide host range, and persistence in soil and water xylem tissues was most conspicuous in Hawaii 7996 (Nakaho for long periods (Genin, 2010; Mansfield et al., 2012). Strong et al., 2004). Other studies reported that the cell walls were quantitative resistance to bacterial wilt in tomato has been thicker in parenchyma and vessel cells of infected xylem tis- available for many decades, but has been successfully deployed sues in resistant LS-89 than in susceptible Ponderosa or mock- only in small-fruited varieties (<200 g) and rootstocks for inoculated plants (Nakaho et al., 2000). Accumulations of grafting, due to a seemingly unbreakable linkage between small electron-dense materials in vessels and parenchyma cells were fruit size and resistance (Scott et al., 2005; Rivard and Louws, also more apparent in LS-89, while Ponderosa showed necrosis 2008). The Hawaii breeding line series, particularly Hawaii in all parenchyma cells adjacent to vessels containing bacteria Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 7996, is the most effective source of resistance against various (Nakaho et al., 2000). A recent microscopic study of the dis- strains of R. solanacearum under different environmental condi- tribution of R. solanacearum in roots of Hawaii 7996 and the tions, and are widely used rootstocks for management of bac- susceptible cultivar West Virginia 700 found that colonization terial wilt (Grimault et al., 1994a; Prior et al., 1996; Wang et al., of the root vascular cylinder was delayed and movement in- 1998). The commercially successful hybrid Shield has been the side the vasculature was spatially restricted in Hawaii 7996 most commonly planted rootstock for bacterial wilt resistance (Caldwell et al., 2017). Together, these studies underscore the in North Carolina, USA, in recent years; in this location, this existence of a complex set of events that restrict bacterial col- hybrid is highly resistant in fields with moderate disease pres- onization in space and time in resistant varieties of tomato. sure (Suchoff et al., 2015) but shows an intermediate resistance However, a systematic investigation of R. solanacearum invasion under strong disease pressure (Kressin, 2018). Resistance in a patterns at the whole-plant and tissue level is lacking. mapping population derived from Hawaii 7996 (resistant) × Here, we applied luminescent and fluorescent bacteria West Virginia 700 (susceptible) has been reported to be mainly for the characterization of resistance to bacterial wilt in to- quantitative, involving two major quantitative trait loci (QTLs) mato root, hypocotyl, and stem organs at the tissue level. We located in chromosomes 12 and 6 (Bwr-12 and Bwr-6), ac- compared highly susceptible, moderately resistant, and highly counting for 18–56% and 11–22% of the phenotypic variation, resistant grafted tomato plants using a standard soil-based seed- respectively (Wang et al., 2013), and three minor loci (Bwr-3, ling grafting method and an in vitro grafting method. We pro- Bwr-4, and Bwr-8). Some of these QTLs are strain- and/or pose an integrative model for bacterial wilt in resistant tomato environment-specific (Thoquet et al., 1996a, b; Mangin et al., lines that highlights the importance of four different ‘bottle- 1999; Wang et al., 2000; Carmeille et al., 2006; Wang et al., necks’ that limit bacterial colonization: (i) invasion of the root; 2013). (ii) vertical movement upwards to the stem; (iii) circular pas- Initial studies of R. solanacearum colonization in several re- sage from vessel to vessel; and (iv) escape from the xylem and sistant and susceptible tomato varieties reported that bacterial radial spread into the pith/cortex tissues. wilt resistance was associated with the capability of the plant to limit bacterial spread from the root collar to the midstem, and not with limited root invasion (Grimault and Prior, 1993; Nakaho, 1997a). However, when similar experiments were per- Materials and methods formed without wounding the roots, limited bacterial growth Plant and bacterial materials and growth conditions in Hawaii 7996 was observed in all tissues analyzed (taproot, The tomato (Solanum lycopersicum) lines used in this study were the highly hypocotyl, petiole and mid-stem; McGarvey et al., 1999). susceptible commercial variety Marmande (Leroy Merlin), the moder- Studies analyzing plant colonization in grafted tomatoes ately resistant commercial hybrid rootstock Shield (Rijk Zwaan), and the showed that the bacterium was capable of crossing the graft highly resistant public open-pollinated breeding line Hawaii 7996. For in vitro experiments, tomato seeds were surface sterilized in 35% junction into the susceptible scion. Hawaii 7996 rootstocks bleach and 0.02% Triton-X 100 for 10 minutes and then rinsed with were the most efficient in limiting susceptible scion infection sterile distilled water five times before sowing them on semi-solid me- to 38% and wilting to only 10% in conditions where suscep- dium [Murashige and Skoog (MS) with agar] in square culture plates tible varieties were 100% infected and wilted (Nakaho et al., (Sudelab S.L.). Plates were placed standing upright in a walk-in tissue 2004). culture growth chamber set at 22 °C under long-day light conditions. For pot experiments, plants were grown on soil (Substrate 2, Klasmann- Microscopic studies of bacterial wilt in tomato described the Deilmann GmbH) mixed with perlite and vermiculite (30:1:1) in a presence of inducible physico-chemical barriers (tyloses, gums, growth chamber: either a FITOCLIMA 1200 (Aralab) set at 27 °C, with and modifications to the primary cell wall) that seemed to LED lighting, or a SCLAB S.L. set at 25 °C, with fluorescence lighting. limit bacterial spread in the resistant variety Caraïbo (Grimault In both cases, conditions were set at 60% humidity and 12 h day/night et al., 1994b). Light microscopic examination of upper hypo- with light intensity of 120–150 µmol·m–2·s–1. All assays were performed using R. solanacearum GMI1000 strain. cotyls revealed that bacterial masses were present only in The constructs PpsbA::LuxCDABE and PpsbA::GFPuv generated the primary xylem tissues of resistant LS-89 plants (derived by Cruz et al. (2014) were naturally transformed into R. solanacearum from the line Hawaii 7998), whereas bacteria were found in GMI1000 to generate the luminescent and fluorescent reporter strains, Restriction of R. solanacearum colonization in resistant tomato | 2159 respectively. R. solanacearum was grown as previously described (Planas- fluorescent lamp (BP330-385 BA420 filter) and a SZX16 stereomicro- Marquès et al., 2018). scope equipped with a DP71 camera system (Olympus). Quantification of mean fluorescence in the green, blue, and red channels was achieved using ImageJ software. Plant grafting For in vitro grafting, seeds were sown on to sterile filter paper placed on MS-containing plates. Eight days after germination (7 for Marmande to Statistical analysis obtain equivalent stem diameters), the cotyledons were removed and the Statistical analyses were performed using Statgraphics software. All statis- plants were cut at a perpendicular angle 1–2 cm below the cotyledons tical tests are indicated in the respective figure legends. using sterile tools. For double-grafted plants, two cuts were made 2–3 cm apart. Rootstocks and scions were transferred to fresh plates without filter paper and matched with the corresponding reciprocal tissues without any stabilizing device. Plates were kept standing upright in the growth Results chamber. After 10 days, successfully healed plants were either pin- inoculated with the luminescent strain of R. solanacearum and monitored The first ‘bottleneck’ in R. solanacearum colonization: over time or transferred to soil-containing pots and grown as described the root-to-shoot boundary for the pathogenicity assays after acclimation for 48 h in transparent boxes Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 (Altuna 2594005, Stewart Garden) with the vented lids opened after 24 h. Limited shoot colonization by R. solanacearum in resistant For standard grafting, plants with stems 1.5–2 mm in diameter (9 days tomatoes has been proposed to be due to reduced spread after sowing) were grafted 2 cm below the cotyledons using a 70° angle of the bacterium from the root to the stem (Grimault and cut and 1.6 or 2 mm diameter grafting clips (Bato Plastics B.V.). Grafted Prior, 1993; Nakaho, 1997a) and/or limited root invasion plants were kept in misted acclimation boxes in growth chambers and ac- in resistant varieties (McGarvey et al., 1999; Caldwell et al., climated to light (24 h darkness, 24 h at 10% light, 24 h at 50% light) and then to ambient humidity (by opening the vents 4 days after grafting and 2017). To clearly define the location within the plant at partly opening the lid for 48 h before removing it). which resistance was acting, we made use of a constitutively luminescent R. solanacearum that we had previously gener- ated (Cruz et al., 2014) to follow bacterial colonization in Plant inoculation and pathogenicity assays resistant and susceptible tomato plants in a non-disruptive For in vitro assays, 10-day-old plantlets or plantlets 10 days after grafting were pin-inoculated 1 cm below the root collar using a sterile 0.3×13 mm manner. For this, we established a miniaturized in vitro needle (30G×½″, BD Microlance, Becton Dickinson) submerged in a 106 tomato–R. solanacearum infection system. Tomato seedlings colony-forming units (CFU)·ml–1 (OD600=0.001) R. solanacearum sus- were grown on semi-solid medium and pin-inoculated in pension. Plates were kept in a growth chamber (25 °C day, 22 °C night), the roots with the luminescent strain.This forced inoculation and wilting symptoms were recorded and bacterial invasion visualized as ensured the infection of all plants to enable the study of bac- described below. For soil-drenching inoculations, plants were grown until they reached terial spread in the plant tissues. Disease symptoms were re- the 7–9 true leaf stage (4–5 weeks after sowing, and 5–6 weeks for grafted corded as the percentage of wilted plants (Fig. 1A) and plants plants). Inoculations were performed by pouring 40 ml of a 107 CFU·ml were photographed using a live imaging system over time. –1 (OD600=0.01) bacterial suspension on every pot after making four This non-destructive assay mimicked the disease symptom- holes in the soil with a disposable 1 ml pipette tip. Infected plants were atology observed in field or greenhouse conditions under kept in a growth chamber set at 27 °C and scored for wilting symptoms using a scale from 0 to 4: 0=healthy plant with no wilt, 1=25%, 2=50%, strong disease pressure, as indicated by the reduced wilting 3=75%, and 4=100% of the canopy wilted. To assess shoot colonization, of the resistant line Hawaii 7996 (H7996) compared with 4–5-week-old plants were pin-inoculated with 10 µl of a 106 CFU·ml–1 the susceptible line Marmande (Fig. 1A). While all tomato suspension (Ishihara et al., 2012) when indicated (see Supplementary Fig. roots in both lines were colonized 3 days post-inoculation S9 at JXB online). (dpi), shoot colonization was clearly delayed and reduced in H7996 compared with Marmande (Fig. 1B). A representa- Assessment of bacterial invasion tive photograph of the assay at 4 dpi, when the susceptible Invasion of tomato tissues by R. solanacearum was assessed using the lu- plants start to wilt, is presented in Fig. 1C. This image shows minescent and fluorescent strains. For in vitro assays, pin-inoculated plants that, besides the difference in shoot colonization in the two were photographed using a live imaging system (ChemiDoc Touch varieties, a colonization ‘bottleneck’ exists in resistant plants Imaging System, Bio-Rad) as previously described by Cruz et al. (2014), at the root collar. In addition, luminescence intensity was using a 5-minute exposure time with 3×3 sensitivity. Images were pro- cessed using Image Lab software (Bio-Rad). Inoculated soil-grown lower in the roots of H7996 (Fig. 1C), indicating lower bac- plants were uprooted and the roots were surface sterilized in water with terial loads compared with Marmande. ~5–10% bleach for at least 1 minute followed by a wash in water. Plants inoculated with the luminescent strain were sliced from the apex to the roots using a sterile razor blade. Transverse sections 1 mm thick and the Resistant rootstocks reduce plant invasion and limit two halves of longitudinal slices 1–2 cm in length were placed flat on a bacterial multiplication in the roots of grafted plants square plate with a misted lid and visualized using a live imaging system as described above. For each location, a 0.5 cm section was excised and To analyze the contribution of the roots to resistance in fur- incubated for at least 30 minutes in a sterile 2 ml tube with 200 µl of ther detail, we grafted rootstocks and scions of Marmande sterile distilled water. Luminescence was measured with a luminometer and H7996. Grafts were made at the upper hypocotyl (FB 12, Berthold Detection Systems). The relative light units per second and at the root collar, and bacterial colonization and dis- (RLU·s–1) were related to CFU·g–1 tissue after dilution plating of samples and CFU counting 24 h later. ease progression were evaluated using the luminescent Plants inoculated with the fluorescent strain were dissected as de- R. solanacearum strain after pin-inoculation of the roots scribed above and photographed using binocular microscopy with a UV (Fig. 2). Resistant H7996 rootstocks hampered bacterial 2160 | Planas-Marquès et al. Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 1. Evaluation of R. solanacearum colonization in in vitro-grown resistant and susceptible tomato plants. Tomato seedlings of the susceptible variety Marmande and the resistant variety Hawaii 7996 (H7996) were pin-inoculated in the roots with a luminescent R. solanacearum strain, and bacterial colonization and wilting symptoms were evaluated over time. (A) Percentage of plants showing wilting symptoms. (B) Percentage of plants colonized in the roots and stems based on the luminescence signal emitted by the reporter strain. (C) Representative photographs showing infected seedlings at 4 days post-inoculation (dpi). The outline of the plants is due to background light from photosynthetic tissues, while luminescence is shown as darker areas. Saturation was never reached. The experiment was repeated three times with similar colonization kinetics; n=20 plants per variety. (This figure is available in colour at JXB online.) colonization of Marmande scions, while Marmande roots To quantify the reduced root colonization with did not prevent colonization of H7996 scions (Fig. 2A). The R. solanacearum in resistant varieties, we measured bacterial presence of a resistant root system was sufficient to cause a loads in the taproot at 3 dpi, when susceptible plants started reduction in shoot colonization, as stem luminescence was to show symptoms. Bacterial concentrations were calculated comparable in grafted plants with or without a resistant from luminescence units (RLU) measured from taproots lower stem (Fig. 2B). with a luminometer, based on the extremely high correlation To strengthen these observations, we investigated root col- (r2=0.96, P<0.0001) between the luminescence emitted by the onization by R. solanacearum in fully developed plants inocu- tissue samples and the bacterial CFU (Supplementary Fig. S3). lated by soil drenching with the luminescent R. solanacearum This experiment revealed that the resistant rootstocks Shield strain. The tomato variety Shield, which is moderately re- and H7996 had a significantly lower mean bacterial density sistant to bacterial wilt, was introduced in these experiments at the root compared with the susceptible variety Marmande, for comparison with the susceptible Marmande and highly re- which exhibited bacterial concentrations two orders of mag- sistant H7996 varieties. We imaged whole roots of plants of nitude higher (Supplementary Fig. S1B). each variety obtained at 6 dpi (see Supplementary Fig. S1 at JXB online), when plants were already showing wilting symp- The second ‘bottleneck’: resistant shoots restrict toms (Supplementary Fig. S2). Roots of Marmande displayed vertical movement of bacteria along the xylem strong luminescence intensity, while roots of Shield or H7996 displayed weak luminescence (Supplementary Fig. S1A). This Next, we investigated R. solanacearum shoot coloniza- phenomenon was consistent regardless of the intensity of the tion in soil-inoculated fully developed Marmande, Shield, signal in the stem or the level of wilting, and correlated with and H7996 plants. Intact 4–5-week-old plants grown in our previous results obtained using the miniaturized in vitro pots could not be imaged for luminescence because of size system (Fig. 1). limitations and reduced sensitivity resulting from the stem Restriction of R. solanacearum colonization in resistant tomato | 2161 Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 2. Bacterial shoot colonization in Marmande and Hawaii 7996 (H7996) grafted plants. Seedlings of Marmande and H7996 were grafted at the mid-stem (A) or the root collar (B) and then pin-inoculated in the roots with the luminescent R. solanacearum strain. A representative photograph of reciprocally grafted plants is shown for each graft type at 10 dpi. The percentages of plants colonized in the roots and the tissues immediately below and above the graft are shown on the right. Arrowheads indicate the grafting junction. Both experiments were repeated at least three times, with similar colonization kinetics. In (A), n=7–8 plants per grafting combination; in (B), n=12–15 plants per grafting combination. (This figure is available in colour at JXB online.) thickness. Therefore, we obtained 1–2 cm stem sections up display a lower number of colonized xylem fiber bundles and to the third internode from plants 6 dpi, when wilting symp- some limited bacterial vertical movement along the vessels, toms could be observed (Supplementary Fig. S2). In order compared with susceptible plants (Fig. 3). to track the movement of luminescent bacteria throughout To avoid between-plant variation in colonization and dir- the stem, slices were taken from the top and bottom of each ectly compare the behavior of susceptible and resistant tissues section and the remaining stem was divided longitudinally when confronted with equivalent bacterial loads, we char- into two pieces. Representative photographs of all sections acterized R. solanacearum distribution in reciprocally grafted from a plant of each variety are presented in Fig. 3. In all plants. We used adult plants inoculated by soil drenching and cases, the luminescence matched the location of xylem bun- monitored the vertical movement of the luminescent bac- dles, indicating that bacteria were mostly confined to the terial strain in the hypocotyl region (where grafting was xylem at this stage of infection. As expected, bacterial colon- performed) at 6 dpi (Fig. 3B). Colonized vessels and lumi- ization in the shoot was much more apparent in Marmande, nescence were almost undetectable in self-grafted resistant as indicated by the intense luminescence observed, compared H7996 (Fig. 3B), similar to what was observed in non- with the resistant varieties, in which luminescence was in grafted plants (Fig. 3A). Self-grafting of the Marmande var- most cases detected only at a higher exposure setting (Fig. 3A iety demonstrated that grafting per se did not restrict vertical Supplementary Fig. S4). In addition, the number of lumi- movement (Fig. 3B). Colonization was hampered in H7996 nescent bundles decreased occasionally with height in the scions grafted on to Marmande rootstocks, and was higher in resistant varieties, whereas it remained constant in the sus- Marmande scions than in the H7996 rootstocks grafted on to ceptible Marmande plants. In summary, resistant tomato lines them (Fig. 3B).These results demonstrated that at comparable 2162 | Planas-Marquès et al. Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 3. Vertical movement of R. solanacearum in tomato shoots. Tomato plants (4–6 weeks old) of (A) the susceptible variety Marmande, the moderately resistant variety Shield, and the highly resistant variety H7996, and (B) reciprocally grafted Marmande and H7996 plants grown in pots were soil inoculated with luminescent R. solanacearum. Shoot sections were obtained at 6 dpi and photographed in a live imager. In (A), photographs show each bisected fragment and its top and bottom slices. Sections were obtained at the base of the hypocotyl, the distal hypocotyl (immediately below the cotyledons), and internodes 1, 2, and 3. In Image Lab software (Bio-Rad) the following ‘High’/‘Low’/‘Gamma’ values were used for low and high exposure settings, respectively: 10000/60/1 and 1300/60/2. In (B), sections were obtained above and below the graft junction. Arrowheads and dotted lines indicate the position of the graft junction. (This figure is available in colour at JXB online.) bacterial concentrations, vertical colonization is inhibited out this possibility, we exchanged a fragment of hypocotyl and overall bacterial density is strongly reduced along the between Marmande and H7996 plants in a double-grafting xylem of H7996 compared with Marmande. Similar results approach (Supplementary Fig. S6). Grafted plants contained were observed in Marmande–Shield grafting combinations a 2 cm fragment of the hypocotyl from one of the varieties (Supplementary Fig. S5). in-between the basal and distal hypocotyl regions of the other A decrease in vertical colonization could be explained by variety (Supplementary Fig. S6A, B). The double-grafted a timing artefact: if luminescence photographs were taken plants were grown on soil to the 7–9 true leaf stage and were too soon for the bacteria to grow in the resistant scion, this infected with the luminescent R. solanacearum strain (Fig. would give a false impression of hampered invasion. To rule S6C–G). As expected, plants that contained the roots and Restriction of R. solanacearum colonization in resistant tomato | 2163 basal hypocotyl from Marmande wilted to a similar extent movement of R. solanacearum along the xylem in a root- to plants with Marmande rootstocks (Supplementary Figs independent manner. S2, S6D, E). We observed and quantified bacterial movement along the xylem in the two combinations of double-grafted Plant wilting is determined by a bacterial density plants using luminescence (Fig. 4). Marmande rootstocks threshold in the hypocotyl were heavily colonized by R. solanacearum, and bacterial density decreased as soon as the pathogen crossed the first To trace the vertical movement of bacteria in the plant in a grafting junction and encountered H7996 tissue. When quantitative manner, we measured bacterial loads from the tap- R. solanacearum moved upwards into susceptible tissue for root to the third internode in at least 30 plants per grafting com- the second time, it again multiplied to high concentrations bination, sampled at different times (3–10 dpi), which showed (Fig. 4). The opposite result was observed in the reciprocal a range of wilting symptoms. The results in Fig. 5 clearly show grafting: colonization with R. solanacearum was hampered in that, regardless of the degree of susceptibility, asymptomatic to- H7996 rootstocks, especially at 10 dpi (Supplementary Fig. mato plants contained bacterial concentrations generally lower S6F, G), increased in Marmande hypocotyls, and decreased than 107 CFU g–1 tissue and wilted plants always showed bac- Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 when the bacteria crossed the second grafting junction and terial counts above this threshold in the taproot and basal hypo- encountered H7996 tissue again (Fig. 4). Altogether, these cotyl, although they may have had lower numbers of bacteria results demonstrate the ability of H7996 to restrict vertical in the shoot above the cotyledons. Additionally, the hypocotyl Fig. 4. Bacterial shoot colonization in Marmande and H7996 double-grafted plants. Seedlings of Marmande and H7996 were double-grafted in the middle of the stem, transferred to pots, and grown for 3–4 weeks. They were then inoculated with the luminescent R. solanacearum strain by soil drenching. (A) Shoot sections from the hypocotyl were obtained at 10 dpi (upper panel) or 23 dpi (lower panel) and photographed in a live imager. ‘Bottom’ and ‘Top’ refer to the basal and distal hypocotyl locations, respectively; ‘Middle’ refers to the region between the two graft junctions. Graft junctions are indicated by arrowheads and dotted lines. (B) Bacterial loads in the shoots of the plants shown in (A) were calculated from the luminescence and are expressed as log CFU g–1 tissue. (This figure is available in colour at JXB online.) 2164 | Planas-Marquès et al. Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 5. Density of colonization of R. solanacearum assessed over the height of grafted tomato plants. Bacterial concentrations at different heights in the tissues of wilting (light grey) and asymptomatic (dark grey) grafted plants are shown. Luminescence was measured with a luminometer in 0.5 cm sections from at least 30 inoculated plants per grafting combination. Bacterial counts were calculated from the luminescence and are expressed as log CFU g–1 tissue. Each dot represents one plant. Only one self-grafted H7996 plant wilted, hence the lack of a boxplot. Values between 0 and 4 lie below the threshold for luminescence detection (see Supplementary Fig. S3) and are considered as 0 here. From left to right, sections correspond to: taproot, basal hypocotyl, distal hypocotyl, and internodes 1, 2, and 3. The dashed line and arrowhead indicate the location of the graft junction. Different letters above each boxplot indicate statistically significant differences (α=0.05, Fisher’s least significant difference test). In each boxplot, the whiskers extend vertically from the hinges (the upper and lower bounds of the box) to the largest (upper whisker) or smallest (lower whisker) value no further than 1.5 × the interquartile range (the distance between the first and third quartiles) from the hinge. Dots beyond the ends of the whiskers are outliers. The horizontal band inside each box indicates the median. (This figure is available in colour at JXB online.) seemed to act as an additional vertical threshold in susceptible H7996, and Shield plants grown in pots with a R. solanacearum plants, since asymptomatic Marmande plants were often col- strain constitutively expressing GFPuv (Cruz et al. 2014) and onized below the hypocotyl but the plants always wilted when observed slices of the shoots with a fluorescence stereomicro- the bacteria moved above the hypocotyl (Fig. 5). In contrast, scope. Fig. 6A shows representative images of transverse hypo- when H7996 scions were grafted on to Marmande rootstocks cotyl sections of the three tomato varieties at 8 dpi. At this —a situation in which the barrier in the roots of the resistant stage, the stem xylem tissue was arranged into four primary variety is absent— the tissues of the resistant variety were able bundles and typically two to four smaller secondary bundles, to cope with high bacterial concentrations in the shoot and the connected by the interfascicular cambium formed by xylem plants remained asymptomatic (Fig. 5). Similar results were ob- parenchyma and some xylary fibers. The microscopic images served using the Shield variety (Supplementary Fig. S7). indicate that R. solanacearum can move horizontally from vessel to vessel (circular movement) and from the vessels to the adja- The third and fourth ‘bottlenecks’: resistant shoots cent parenchyma tissues (radial movement). In the susceptible restrict circular and radial movement of bacteria variety Marmande, fluorescent bacteria occupied almost the entire vascular ring and even extended radially to the apoplast In order to examine the patterns of colonization within the of the pith and cortical tissues (Fig. 6A). In contrast, in the re- stems at the tissue level, we inoculated 4-week-old Marmande, sistant variety H7996 bacteria were confined to a few single Restriction of R. solanacearum colonization in resistant tomato | 2165 Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 6. Distribution of a fluorescent R. solanacearum strain in susceptible and resistant tomato shoots. (A) Plants (4–5 weeks old) of the susceptible variety Marmande, the moderately resistant variety Shield, and the highly resistant variety H7996 grown in pots were inoculated with a fluorescent R. solanacearum strain by soil drenching. Basal hypocotyl stem sections were obtained and photographed with a fluorescence stereomicroscope under white light (upper panels) and UV light (middle and lower panels). The lower panels show a magnification of the areas indicated with rectangles in the middle panels. The sections were visualized through a UV light filter, highlighting the autofluorescence of lignin in blue and the fluorescence emitted by the bacteria in green. Green dots correspond to clumps of bacteria. Arrowheads indicate xylem vessels with limited colonization. (B) Plants consisting of H7996 scions grafted on to Marmande rootstocks were grown and inoculated with the fluorescent strain, and transverse sections taken at different heights below and above the graft junction were photographed with a fluorescence stereomicroscope. (C) Fluorescence photomicrographs of highly colonized and fully wilted Marmande and H7996 shoots from four plants at the basal hypocotyl and the first internode. xylem vessels (Fig. 6A).The moderately resistant variety Shield rootstocks, to enable high bacterial numbers to reach the re- showed an intermediate phenotype, with colonization more sistant stem tissues (Figs 3B, 4, 5), and inoculated the grafted restricted to the vascular ring and limited radial spread to plants with the fluorescent R. solanacearum strain by soil neighboring tissues (Fig. 6A). drenching. Examination of shoot sections obtained at different The extremely limited vertical colonization of the xylem shoot heights at 8 dpi revealed extensive vertical, circular, and in H7996 hampered precise characterization of the circular radial colonization of the Marmande tissues below the graft and radial bacterial movements in the shoots. To overcome (Fig. 6B, Supplementary Fig. S8).The section at the graft junc- this limitation, we grafted H7996 scions on to Marmande tion showed that the H7996 tissues immediately blocked the 2166 | Planas-Marquès et al. spread of the bacterium both circularly through the xylem ring the resistance observed in controlled-environment studies with and radially to the pith and cortical tissues (Fig. 6B). These re- comparable conditions and plant ages for the different var- strictions became more apparent in higher sections consisting ieties investigated (Supplementary Fig. S2; Wang et al., 1998; exclusively of resistant tissue (Fig. 6B, Supplementary Fig. S8). McGarvey, Denny and Schell, 1999; Nakaho et al. 2004; Rivard To better compare the behavior of R. solanacearum in re- and Louws, 2008). Root pin-inoculation of plantlets grown in sistant and susceptible tissues, we repeated this experiment vitro showed similar results (Fig. 1A), but bacterial concentra- using a larger number of plants, and observed shoot sections tions reached higher numbers in the tissues of pin-inoculated of resistant scions that showed the most pronounced wilting resistant varieties compared with soil-drenched plants of the with the fluorescence stereomicroscope. Fig. 6C shows same resistant varieties, while the susceptible variety was these H7996 shoot sections confronted with a high bac- highly colonized by both methods of inoculation (Figs. 1 and terial inoculum introduced from the susceptible rootstock, 2, Supplementary Fig. S1). The differences in the inoculation compared with Marmande shoot sections. Notably, radial methods imply that resistant varieties have the ability to re- movement of bacteria from the highly colonized xylem strict invasion of the root—a step that is overcome when pin- bundles was strongly restricted in the H7996 shoots, even inoculation of the roots is used-. Our findings are in agreement Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 when the xylem tissue was highly colonized (Fig. 6C right with the limited bacterial growth in the taproot of H7996 panel). This restriction was also observed when the fluores- observed when roots were not wounded before inoculation cent R. solanacearum strain was directly pin-inoculated into (McGarvey et al., 1999). Additionally, in vitro-grafted pin- the shoots (Supplementary Fig. S9). inoculated plants displayed slightly delayed colonization com- Finally, we performed a time-course invasion assay in which pared with non-grafted plants (3 dpi in Fig. 1A versus 5 dpi in we quantified the amounts of bacteria that were moving out- Fig. 2). This difference might be linked to the developmental side the vascular ring over time (Fig. 7). We observed that stage. Since older plants (in this case the grafted ones) are more R. solanacearum escaped from the vascular ring as early as developed, their cell walls might be reinforced, thus partly 5 dpi and heavily colonized the pith and cortical tissues of hindering R. solanacearum invasion. Finally, the pin-inoculated Marmande by 9 dpi (Fig. 7). Moreover, the amount of bac- resistant plants that are highly colonized likely mimic the situ- teria outside the vascular tissues was directly correlated with ation encountered in nature when environmental conditions the extent of vascular ring colonization (Fig. 7B). This con- are highly favorable to the pathogen. Indeed, it has been shown trasted with the ability of H7996 shoots to impede the escape that even the most highly resistant varieties that are available of the pathogen from the vascular ring (Figs 6 and 7). These cannot completely prevent root and stem colonization by results indicate that the capacity of R. solanacearum to radially R. solanacearum in greenhouse conditions (Nakaho, 1997a, b; invade the pith and cortex tissues is dependent on the degree Nakaho et al., 2004). of susceptibility of the plant and that invasion occurs as a con- sequence of increased colonization. Restriction of vertical movement up the stem The fact that R. solanacearum can colonize the stems of many resistant tomato plants when soil-drenching is used as the Discussion method of inoculation indicates that additional mechanisms In this work we propose a model that relates the spatio-temporal of resistance must be in place in the aerial tissues to prevent dynamics of R. solanacearum invasion and proliferation in to- wilting. Previous studies have demonstrated that bacterial mato plants to disease development and shows how quanti- counts in the stems of resistant tomato plants were always lower tative resistance affects these parameters (Fig. 8). Systematic than those in susceptible varieties and that this was due to a analysis of the progression of bacterial colonization inside the limitation of pathogen movement from the primary xylem to plant reveals four clear limitations of growth in resistant to- other xylem tissues (Nakaho et al., 2004). In this work, we have mato tissues that hamper disease progression: colonization of analyzed the vertical dimension of bacterial spread and dem- the root, invasion of the stem vascular bundle, vertical inva- onstrated that resistant tissues limit movement up the xylem sion up the vessels, and invasion of the pith/cortex. The ba- vessels (Fig. 3). Double-grafting experiments, in which a small sically binary outcome of death-by-permanent-wilting caused portion of resistant stem was introduced into an otherwise sus- by R. solanacearum in tomato seems to require the bacterium ceptible adult plant or vice versa, ruled out any effect of grafting to surmount each of these physio-anatomical plant barriers, per se on bacterial movement inside the xylem and suggested which are quantitatively determined by host resistance.We will that resistance to bacterial wilt could be due to non-diffusible discuss each of these four important ’bottlenecks’ that can de- xylem structures/compounds originating from the stem, as has termine either host resistance or successful colonization of the been described for other bacterial vascular diseases (Chatterjee plant by the bacterium. et al., 2008). The nature of the plant components or structures hindering Restriction of root colonization root-to-shoot vertical bacterial movement is still unknown, al- though reports have described the presence of tyloses (evagin- We analyzed the interaction of R. solanacearum with tomato ations of the adjacent parenchyma cells into the xylem lumen) using two main variables: susceptible versus resistant varieties and gums that seemed to limit bacterial spread in the xylem of and soil drenching versus pin-inoculation. Soil-drenching in- bacterial wilt-resistant Caraïbo tomato plants (Grimault et al., oculations clearly reproduced the progression of disease and 1994b). Obstruction of xylem vessels with gums and tyloses Restriction of R. solanacearum colonization in resistant tomato | 2167 Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 7. Time course of invasion of the fluorescent R. solanacearum strain in grafted tomato shoots. (A) Fluorescence photomicrographs of self-grafted Marmande and plants consisting of H7996 scions grafted on to Marmande rootstocks inoculated with the fluorescent R. solanacearum strain. Sections were taken at the basal hypocotyl and the first internode. The sections were visualized through a UV light filter, highlighting the autofluorescence of lignin in blue and the fluorescence emitted by the bacteria in green. (B) Quantification of fluorescence signal (AU, arbitrary units) within and outside the vascular ring in the basal hypocotyl and the first internode of plants from each stage of the infection shown in (A). Three biological replicates (n=3) were used. Data presented are means ± SE. Different letters above the bars indicate statistically significant differences (α=0.05, Fisher’s least significant difference test). is a common plant response to restrict systemic infection by vascular gelation is considered an essential part of Fusarium wilt vascular pathogens (VanderMolen et al., 1987; Grimault et al., resistance in carnation (Baayen and Elgersma, 1985). Tyloses 1994; Clérivet et al., 2000; Sun et al., 2013). For instance, have been similarly proposed to restrict pathogen movement 2168 | Planas-Marquès et al. Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 Fig. 8. Model of the tomato–R. solanacearum pathosystem in susceptible and resistant germplasm. Schematic representation of the colonization movements of R. solanacearum (shown in green) in susceptible and resistant tomato tissues. in tomato cultivars resistant to the vascular pathogens Fusarium susceptibility has been observed in potato (Cruz et al., 2014; oxysporum, Verticillium abo-atrum, and R. solanacearum (Hutson Ferreira et al., 2017). Similarly, X. fastidiosa has been shown to and Smith, 1980; VanderMolen et al., 1987; Grimault et al., invade 10 times fewer stem vessels and exhibit lower population 1994b). Although Grimault et al. (1994b) correlated the pres- densities in resistant grapevine cultivars (Chatterjee et al., 2008). ence of tyloses in Caraïbo to the limitation of R. solanacearum These results indicate that limitation of circular movement in spread, in another resistant cultivar (LS-89) the formation of the xylem ring is a conserved mechanism for resistance against these structures was neither induced by the pathogen nor vascular bacterial pathogens. Restriction of R. solanacearum seemed to affect bacterial colonization (Nakaho, 1997a). to the primary xylem vessels could explain why resistant to- Similarly, tyloses formed in grapevines in response to Xylella mato plants often remain asymptomatic. If a blockage occurs fastidiosa infection were found to be more abundant in sus- in the primary xylem, which is largely non-functional after ceptible cultivars and did not affect the vertical movement of the secondary xylem has been produced (Esau, 1977), flow the pathogen (Sun et al., 2013).These observations suggest that conduction could occur undisturbed through the uninfected the role of tyloses in vascular pathogen restriction may be cul- secondary xylem. tivar- or species-specific and/or depend on the lignification In susceptible plants, R. solanacearum can move horizontally status of the plant host. The results presented here and our through the xylem ring by directly degrading the cell walls recent finding that R. solanacearum-tolerant potato lines also of primary xylem vessels or pit membranes in the secondary induced the development of tyloses upon infection (Ferreira xylem vessels (Wallis and Truter, 1978; Grimault et al., 1994b; et al., 2017) seem to indicate that these structures are important Vasse et al., 1995; Nakaho et al., 2000). To counter such cir- components of bacterial wilt resistance in solanaceous plants. cular movement, plants have evolved structural defenses that are induced upon attack by vascular pathogens, involving the Restriction of vascular bundle invasion and the deposition of various coating materials to reinforce the walls bacterial density threshold of xylem vessels, pit membranes, and surrounding parenchyma cells. Vascular coatings are thicker in resistant tomato cultivars Restriction of R. solanacearum colonization in the stems of infected with R. solanacearum, and this may be the cause of H7996 is also achieved by limiting the horizontal movement the observed limitation of bacterial movement between xylem of the pathogen from vessel to vessel (referred to hereafter as tissues (Nakaho et al., 2000, 2004). The detailed description circular movement). Confinement of R. solanacearum to pri- of the process we present here will be crucial to decipher the mary xylem vessels has been observed in the stems and roots genetic determinants and the composition of these vascular of different resistant tomato cultivars compared with suscep- coatings, which remain unknown. tible ones (Nakaho, 1997a; Nakaho et al., 2004; Caldwell et al., Restriction of circular movement in the stem is a very ef- 2017). A similar correlation between R. solanacearum move- ficient confinement strategy, since it is still acting when ment between stem vessels, bacterial growth, and the level of high loads of bacteria are forced into the stem through root Restriction of R. solanacearum colonization in resistant tomato | 2169 inoculations using H7996 scions grafted on to Marmande to access resistant tissues, as can be seen in some of the images rootstocks (Fig. 6). However, there seems to be an upper in Fig. 6C. This observation is in agreement with a previous limit of bacterial inoculum above which this restriction is no report showing that when high bacterial inocula (109 CFU longer effective (see plant 4 in the lower panel of Fig. 6C). ml–1) were used, R. solanacearum could also be detected in the This finding is in agreement with previous reports showing parenchyma cells of resistant tomato (Nakaho, 1997b). Thus, that delivery of a high R. solanacearum inoculum (109 CFU restriction of radial bacterial movement is no longer effective ml–1) directly into tomato stems overcomes resistance (Nakaho, when the bacterial density exceeds a certain threshold. 1997b). This idea relates to the concept of a density threshold Structural changes in cell walls and pit membranes in re- in the interaction between tomato and R. solanacearum. Earlier sponse to R. solanacearum infection are more conspicuous in observations established the onset of bacterial wilt symptoms resistant tomato (Nakaho et al., 2000). Thus, bacteria may be at a bacterial density in the stem of between 106 and 108 CFU prevented from escaping the xylem in resistant tomato by g–1 fresh tissue (Grimault and Prior, 1994; Nakaho, 1997a; a combination of inducible structural defense mechanisms Huang and Allen, 2000; Nakaho et al., 2004). We have char- that may appear later and/or with less intensity in susceptible acterized this threshold systematically by assessing bacterial lines, rendering them ineffective to restrict colonization. Downloaded from https://academic.oup.com/jxb/article-abstract/71/6/2157/5686149 by guest on 03 June 2020 densities throughout the plant in large populations of grafted Interestingly, slightly decreased invasion was also observed tomato plants with varying resistance. We conclude that, in in the susceptible hypocotyls of the Marmande–H7996 both resistant and susceptible varieties, symptoms invariably grafting combination (Fig. 7). This finding could be ex- appear when bacterial populations in the hypocotyl exceed plained by a cross-talk between the scion and rootstock. This a threshold of 107 CFU g–1 tissue (Fig. 5, Supplementary Fig. interaction could trigger the expression of putative defense- S7). Plating dilutions of homogenized tissues to establish bac- related genes or genes that reinforce plant cell wall structures terial counts is labor intensive, but we show that light emission in the susceptible rootstock. Alternatively, defense-related or from tissues inoculated with a luminescent strain is a useful structure-remodeling proteins might be secreted by the re- alternative measure of bacterial counts (correlation coefficient sistant scion and reinforce nearby tissues (in this case the sus- r2=0.96). Since bacterial density and distribution are predictive ceptible hypocotyl). These two explanations seem plausible of the degree of disease resistance, we have started to use lu- given the existing vascular connectivity between the grafted minescent strains to screen potato germplasm for resistance to tissues. Indeed, transcriptional reprogramming occurs even bacterial wilt as a way of aiding the breeding process (Cruz in rootstocks and scions of the tomato/potato heterografting et al., 2014; Ferreira et al., 2017). system (Zhang et al., 2019). Additionally, peroxidases and other cell wall remodeling enzymes, such as glycosyl Restriction of radial movement out of the xylem into hydrolases, are secreted into the xylem by the resistant line the pith and cortex H7996 upon R. solanacearum infection (M. Planas-Marquès, F. Kaschani, M. Kaiser, R. A. L. van der Hoorn, M. Valls, Our work has also revealed a fourth limitation restricting bac- N. Sánchez-Coll, unpublished results). Hence, increased lig- terial spread in the tissues of resistant tomato varieties: restric- nification and cell wall reinforcement could also take place tion of radial movement of R. solanacearum out of the xylem in neighboring susceptible tissues in grafted plants. into the adjacent parenchyma cells in the pith and cortex (Figs 6 and 7).These metabolically active cells are in close con- An integrated model for tomato resistance to tact with the xylem vessels through the pits and are thought bacterial wilt to be pivotal for the induction of plant defenses against patho- gens within the xylem, although very little is known about the As we have discussed, in the past three decades, various labora- mechanisms regulating this response. tories have aimed to understand how resistant tomato varieties Earlier studies detected widespread R. solanacearum colon- restrict R. solanacearum colonization and remain asymptomatic ization of stem parenchyma cells in susceptible tomato var- despite having relatively high bacterial loads. The fact that the ieties at late stages of infection, when plants showed extensive defenses are not limited to a particular plant site, as the bac- wilting (Nakaho, 1997a; Nakaho et al., 2000). These cells ap- terium has to traverse different tissues to reach the xylem, has peared to be filled with bacteria and displayed necrosis and complicated this work. A question that arises is whether the signs of degeneration. In contrast, in resistant tomato varieties, xylem is the final destination of R. solanacearum. necrotic parenchyma cells containing bacteria were observed Here, we have defined the barriers encountered by only occasionally (Nakaho et al., 2000). Our data confirm R. solanacearum as it progresses from the soil into the xylem these observations and additionally show that parenchyma cell and have found that, after systematic spread through the xylem, invasion starts at early times in susceptible plants (5 dpi in our the final destination of the bacterium may be extensive inva- experimental setup) and spreads massively through the pith at sion of the stem apoplast. It has already been suggested that later time points (8–9 dpi, Fig. 7A). In contrast, colonization vascular bacteria use plant cell wall degradation products as remains limited to xylem vessels in resistant tomato (Fig. 6). carbon and energy sources (Chatterjee et al., 2008; Genin and As was the case for the circular bacterial movements de- Denny, 2012). It is tempting to speculate that R. solanacearum scribed above, radial restriction of movement out of the xylem has evolved not only to colonize the xylem but also to escape in resistant varieties could be partially overridden by grafting from it to obtain richer sources of nutrition from metabolic- to susceptible rootstocks that enabled high bacterial densities ally active parenchyma cells, facilitating the death and decay of 2170 | Planas-Marquès et al. infected plants and thus allowing the pathogen to spread in the References soil and move into the next host. Baayen RP, Elgersma DM. 1985. Colonization and histopathology of sus- In conclusion, here we clearly define four ‘bottlenecks’ to ceptible and resistant carnation cultivars infected with Fusarium oxysporum bacterial colonization in tomato and demonstrate that the de- f. sp. dianthi. 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