The Plant Journal (2022) 110, 452–469 doi: 10.1111/tpj.15680 Phosphate-induced resistance to pathogen infection in Arabidopsis Beatriz Val-Torregrosa1 , Mireia Bundo  1 , Hector Martın-Cardoso1 , Marcel Bach-Pages1 , Tzyy-Jen Chiou2 , Victor Flors3 and Blanca San Segundo * 1,4 , 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus Universitat Auto noma de Barcelona (UAB), Bellaterra (Cerdanyola del Valle s), Barcelona, Spain, 2 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, 3 Departamento de Ciencias Agrarias y del Medio Natural, Escuela Superior de Tecnologıa y Ciencias Experimentales, Universitat Jaume I, Castello , Spain, and 4 Consejo Superior de Investigaciones Cientıficas (CSIC), Barcelona, Spain Received 13 February 2021; revised 30 December 2021; accepted 17 January 2022; published online 21 January 2022 *For correspondence (e-mail blanca.sansegundo@cragenomica.es). SUMMARY In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudi- mentary. Here we show that MIR399 overexpression, loss-of-function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibio- trophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the sali- cylic acid (SA)- and jasmonic acid (JA)-dependent signaling pathways were higher in high Pi plants compared to wild-type plants grown under control conditions, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infec- tion by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses. Keywords: Arabidopsis thaliana, Colletotrichum higginsianum, immune response, jasmonic acid (JA), mi- croRNA399 (miR399), phosphate (Pi), PHOSPHATE 2, Plectosphaerella cucumerina, reactive oxygen species (ROS), salicylic acid (SA). INTRODUCTION et al., 2013; Snoeijers et al., 2000). However, the effect of In nature, plants are simultaneously exposed to a combina- combined abiotic and biotic stress factors might vary tion of biotic and abiotic stresses that are diverse in time depending on the nature of these interactions, and the and space, which requires proper integration and interac- plant response to simultaneously or sequentially applied tions between different stress response pathways. Expo- stresses cannot be simply inferred from responses to indi- sure to a single stress might, however, impact the plant vidual stresses (Coolen et al., 2016; Nobori & Tsuda, 2019; response to another stress (Kissoudis et al., 2014; Nejat & Pandey et al., 2017; Prasch & Sonnewald, 2013). As stress Mantri, 2017; Pandey et al., 2017). For instance, plant responses are costly, when facing multiple stresses simul- immune responses and disease resistance can be altered taneously, plants need to prioritize their stress responses in plants exposed to drought or high salinity (Atkinson & for efficient use of finite resources, in accordance with the Urwin, 2012; Bostock et al., 2014; Yasuda et al., 2008). optimal defense theory (ODT) (Meldau et al., 2012). Inappropriate supply of mineral nutrients (e.g., nitrogen According to the ODT, stress responses are prioritized in supply) might also impact disease severity (Ballini the most valuable parts (Keith & Mitchell-Olds, 2017), and 452 Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Phosphate-induced disease resistance in Arabidopsis 453 recent findings indicate that Arabidopsis plants spatially organismal level, interactions between hormone signaling separate contrasting stress responses in leaves of different pathways might also balance trade-offs between conflict- ages (e.g., young leaves exhibit higher biotic stress ing biotic and abiotic stress responses (i.e., prioritization of responses but lower abiotic stress responses compared responses in leaves of Arabidopsis plants) (Berens with old leaves) (Berens et al., 2019; Wolinska & et al., 2019; Wolinska & Berens, 2019). Sugar and ROS Berens, 2019). To date, little information is available on the have also been proposed as candidate signaling molecules molecular mechanisms by which biotic and abiotic stress to regulate prioritization between biotic and abiotic stress responses are differentially prioritized in plants and how responses (Wolinska & Berens, 2019). they adapt to conflicting stresses for optimal responses. Plant microRNAs (miRNAs) are a class of small RNA To defend themselves against pathogens, plants have molecules that mediate post-transcriptional gene silencing evolved an innate immune system in which many intercon- through sequence complementarity with cognate target nected processes are involved (Jones & Dangl, 2006). transcripts (Bartel, 2004; Xie et al., 2005). They are tran- Pathogen-induced pathways are defined principally accord- scribed from MIR genes as long precursor transcripts that ing to the molecules recognized by the host plant (Boller & adopt a stem-loop structure by self-complementarity that Felix, 2009; Couto & Zipfel, 2016; Jones & Dangl, 2006; are processed by a RNase III DICER-like, typically DCL1, to Thomma et al., 2011; Upson et al., 2018). Plants recognize produce a double stranded duplex, the miRNA-5p/miRNA- pathogen-associated molecular patterns (PAMPs) by recep- 3p duplex (previously named as miRNA/miRNA*) (Borges & tors at the plasma membrane, which triggers the induction Martienssen, 2015). The functional strand of the duplex is of multiple cascades leading to the induction of immune selectively loaded into an ARGONAUTE protein, the effector responses, referred to as PAMP-triggered immunity (PTI). protein of the RNA-induced silencing complex (RISC), which Components of PTI include the reinforcement of cell walls, mediates post-transcriptional gene silencing by cleaving the accumulation of reactive oxygen species (ROS), activation target transcripts or by translational inhibition (Brodersen of phosphorylation cascades, production of antimicrobial et al., 2008; Llave et al., 2002). The important role of plant compounds, and accumulation of pathogenesis-related miRNAs in diverse developmental processes and adaptation proteins, among others (Andersen et al., 2018). Some suc- to environmental stresses is well documented (Chen, 2009; cessful pathogens can overcome PTI by delivering effec- Seo et al., 2013; Song et al., 2019; Staiger et al., 2013). tors into plant cells that suppress PTI, thus leading to The first plant miRNA demonstrated to be involved in disease susceptibility. In turn, some plants have evolved immunity was the Arabidopsis miR393. Here, perception of another immune response in which microbial effectors (or the elicitor flg22 induces miR393 accumulation and down- host proteins modified by effectors) are recognized by regulation of auxin receptors, resulting in resistance to intracellular receptor proteins encoded by resistance genes bacterial pathogens (Navarro et al., 2006). Since then, (Han, 2019). This recognition triggers a rapid and robust other miRNAs controlling diverse processes have been defense response, the so-called effector-triggered immu- shown to be involved in Arabidopsis immunity, either ETI nity (ETI) (Jones & Dangl, 2006). ETI is often accompanied or PTI (Huang et al., 2016; Jagadeeswaran et al., 2009; Seo by a hypersensitive response (HR) at the infection site, a et al., 2013; Shivaprasad et al., 2012; Staiger et al., 2013). form of programmed cell death (Thakur et al., 2019). How- Some examples are miR160, miR396, miR398, miR400, ever, some PAMPs (e.g., the bacterial Harpin HrpZ protein) miR472, miR773, miR844, miR858, miR863, and miR156 can also induce HR in plants (Chang & Nick, 2012). (Boccara et al., 2014; Camargo-Ramırez et al., 2017; Lee Among ROS, H2O2 is relatively stable and is an impor- et al., 2015; Li et al., 2010; Niu et al., 2016; Park tant molecule regulating plant immunity (Torres et al., 2014; Salvador-Guirao et al., 2018; Yin et al., 2019). et al., 2006). H2O2 might have a direct antimicrobial role Depending on their target gene, these miRNAs can func- against the invading pathogen and also provokes cross- tion as positive or negative regulators in fine-tuning linking of cell wall components to arrest pathogen inva- immune responses. Even though pathogen infection has sion. ROS also function as molecules for the activation of been shown to induce alterations in the expression of a defense mechanisms, and triggers localized cell death plethora of Arabidopsis miRNAs, the mechanistic role of around the infection site (Torres et al., 2002). Phytohor- these miRNAs in processes underling immunity is often mones, together with ROS, provide important signals to unclear. Additionally, most studies on miRNAs involved in help orchestrate plant responses to abiotic and biotic stres- plant immunity have been carried out in the interaction of ses. Immune responses are largely coordinated by the phy- Arabidopsis plants with the bacterial pathogen Pseu- tohormones salicylic acid (SA), ethylene (ET), and domonas syringae, and less is known on miRNAs involved jasmonic acid (JA) (Aerts et al., 2021). Plant hormones do in resistance against fungal pathogens. not function independently, but synergistic or antagonistic On the other hand, distinct miRNAs have been associ- interactions between hormone pathways ultimately drive ated with regulation of nutrient homeostasis in plants (Paul the fine-tuning of plant defense responses. At the et al., 2015). Perhaps the best-known example is miR399, Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 454 Beatriz Val-Torregrosa et al. which is involved in the control of phosphate (Pi) home- investigated whether miR399f overexpression, and subse- ostasis in Arabidopsis plants (Chiou et al., 2006). Under quent Pi accumulation, has an effect on resistance to limiting Pi conditions, miR399 accumulation increases and pathogen infection in Arabidopsis. To this end, transgenic causes repression of its target gene, PHOSPHATE2 (PHO2), plants overexpressing MIR399f (hereinafter referred to as encoding an E2 ubiquitin-conjugating enzyme responsible miR399 OE plants) were generated. Compared to wild-type for degradation of phosphate transporters (Chiou plants, miR399 OE lines accumulated precursor and mature et al., 2006; Fujii et al., 2005; Huang et al., 2013; Kraft miR399 sequences. The observed increase in miR399 abun- et al., 2016; Liu et al., 2012). Hence, miR399 accumulation dance in miR399 OE lines was accompanied by a decrease in response to Pi starvation relieves negative post- in PHO2 transcript level and overaccumulation of Pi in transcriptional control of Pi transporters and promotes rosette leaves (Figure 1a). uptake of Pi in Arabidopsis plants. The miR399/PHO2 regu- The miR399 OE lines were challenged with the fungus P. latory module has been also recognized as a regulator of cucumerina, the causal agent of the sudden death and Pi homeostasis in rice (Oryza sativa) (Chien et al., 2017; blight disease in many dicotyledonous species. The Ara- Puga et al., 2017). bidopsis–P. cucumerina pathosystem is a widely used In this work, we investigated whether miR399 plays a model system for studies on disease resistance against role in disease resistance in Arabidopsis. We show that necrotrophic fungi (Sanchez-Vallet et al., 2012; Ton & miR399 overexpression and loss-of-function of PHO2 Mauch-Mani, 2004). At the time of pathogen inoculation (3- causes an increase in Pi levels, these plants also exhibiting week-old plants), no obvious phenotypic differences were resistance to infection by necrotrophic (Plectosphaerella observed between miR399 OE plants and wild-type plants cucumerina) and hemibiotrophic (Colletotrichum higgin- (Figure S1a,b). However, at a later developmental stage, sianum) fungal pathogens. Growing Arabidopsis plants the miR399 and pho2 plants displayed symptoms of Pi under high Pi supply also increases resistance to infection excess (e.g., chlorosis on mature leaves of adult plants), by these pathogens. In the absence of pathogen infection, most probably, because of Pi accumulation overtime (re- plants that overaccumulate Pi (e.g., miR399 overexpres- sults not shown; similar results were previously reported sors, pho2 mutants, and wild-type plants grown under by Chiou et al., 2006). high Pi supply) showed ROS accumulation, increased SA Upon pathogen challenge, wild-type plants were and JA levels, and upregulation of genes involved in SA- severely affected by P. cucumerina, while the miR399 OE and JA-dependent defense pathways. Pathogen infection plants consistently exhibited enhanced resistance (Fig- was found to be associated with a higher production of ure 1b). While 75–85% of miR399 OE plants survived at ROS in high Pi plants. Of note, during pathogen infection, 7 dpi, only 20% of the wild-type plants were able to over- induction of the ERF1 branch of the JA pathway and come infection (Figure 1c, left panel). Quantitative PCR repression of the MYC2/VSP2 branch occur in high Pi (qPCR) measurement of fungal DNA confirmed that less plants. Overall, our results support the notion that an fungal biomass was present in leaves of miR399 OE plants increase in Pi content has an impact on hormone networks compared with leaves of wild-type plants infected with P. regulating Arabidopsis defense and promotes resistance to cucumerina (Figure 1c, right panel), which is consistent pathogen infection in Arabidopsis. These results are mark- with the phenotype of resistance that is observed in edly different from those recently reported in rice miR399 OE plants. (Campos-Soriano et al., 2020), where miR399 overexpres- Trypan blue staining was used to visualize both fungal sion and high Pi supply were found to enhance susceptibil- structures and dead cells in the fungal-infected leaves of ity to infection by the rice blast fungus Magnaporthe wild-type and miR399 OE plants. Whereas extensive fungal oryzae. These findings illustrate the need of investigating growth occurred in leaves of wild-type plants, no fungal the effects of nutrient supply on the expression of immune growth could be observed in leaves of miR399 OE plants responses and disease resistance on a case-by-case basis. (Figure 1d). Instead, scattered groups of dead cells were visualized in P. cucumerina-infected miR399 OE plants (Fig- RESULTS ure 1d, right panels). Since P. cucumerina is a necrotrophic fungus, we Resistance to infection by fungal pathogens in hypothesized that the effect of miR399 overexpression on Arabidopsis plants overexpressing MIR399 disease resistance might be dependent on the lifestyle of In Arabidopsis, the miR399 family comprises six members, this pathogen. Accordingly, we investigated resistance of MIR399a–f. Of these, miR399f has identical mature miR399 OE plants to infection by the hemibiotrophic fun- sequences in Arabidopsis and rice. Previous studies indi- gus C. higginsianum. This fungus causes the anthracnose cated that transgenic Arabidopsis and rice plants overex- leaf spot disease on Brassica species, as well as A. thaliana pressing miR399f overaccumulate Pi in leaves (Campos- (O’Connell et al., 2004). The miR399 OE plants showed Soriano et al., 2020; Chiou et al., 2006). In this work, we resistance to C. higginsianum infection relative to wild- Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 455 Figure 1. Resistance of miR399 OE plants to infec- (a) tion by the necrotrophic fungus P. cucumerina. Relative expression Homozygous miR399 OE lines (#1, #4, and #10) and wild-type plants were grown in soil for 3 weeks and then assayed for disease resistance. Three indepen- dent experiments were carried out with at least 12 plants per line in each experiment. (a) Accumula- tion of precursor (pre-miR399) and mature miR399 sequences was determined by RT-qPCR and stem- loop RT-qPCR, respectively. Expression of the miR399 target AtPHO2 was determined by RT- qPCR. The Arabidopsis b-tubulin2 gene (At5g05620) was used to normalize transcript levels (relative (b) P. cucumerina (c) expression). The accumulation of free Pi in leaves is shown (right panel). Bars represent mean  SEM of 100 ** three biological replicates with at least three plants ** 80 per replicate (t-test, *P ≤ 0.05, **P ≤ 0.01, ** ***P ≤ 0.001). (b) Plants were spray-inoculated with 60 P. cucumerina spores (5 9 105 spores ml1). Pic- tures were taken at 7 days post-inoculation (dpi). (c) WT miR399 OE #1 40 Survival ratio of wild-type and miR399 OE plants at 20 7 dpi. Quantification of P. cucumerina DNA was performed by qPCR using specific primers for P. 0 cucumerina b-tubulin at 7 dpi. Values of fungal WT #1 #4 #10 DNA were normalized against the Arabidopsis UBI- miR399 OE QUITIN21 gene (At5g25760). Comparisons have miR399 OE #4 miR399 OE #10 been made relative to wild-type plants. Data are mean  SEM (n = 7) (t-test, *P ≤ 0.05, **P ≤ 0.01). (d) Trypan blue staining of P. cucumerina-infected (d) leaves of wild-type and miR399 OE plants (7 dpi). h, WT miR399 OE hyphae. Arrows and arrowheads indicate fungal hyphae and dead cells, respectively. Higher magni- fications are shown (wild type and miR399 OE, right h panels). Bars represent 300 lm. h type plants, as revealed by quantification of diseased leaf plants in that they both show Pi overaccumulation in area and fungal biomass (Figure 2a). As observed in infec- leaves resulting from increased Pi uptake from roots and tion assays with P. cucumerina, the C. higginsianum-in- root-to-shoot translocation (Aung et al., 2006) (Figure S2b). fected miR399 OE plants exhibited groups of dead cells in We therefore hypothesized that loss-of-function of PHO2 their leaves (Figure 2b). might result in a similar disease phenotype as suppression Collectively, these results demonstrate that miR399 over- of PHO2 by overexpression of miR399. expression enhances resistance to infection by necro- The pho2 plants were then examined for pathogen resis- trophic (P. cucumerina) and hemibiotrophic (C. tance. No phenotypic differences were observed between higginsianum) fungal pathogens. Hence, it is likely that pho2 and wild-type plants at the time of inoculation (Fig- miR399-mediated resistance in Arabidopsis does not ure S2c). Consistent with the disease phenotype observed depend on the lifestyle of the fungus. A pattern of cell in miR399 OE plants, the pho2 mutant exhibited resistance death occurs in the fungal-infected miR399 OE plants. to infection by P. cucumerina (Figure 3a), which was con- firmed by quantifying the survival ratio of the infected pho2 mutant plants exhibit resistance to infection by plants and the amount of fungal biomass (Figure 3b). Try- fungal pathogens pan blue staining revealed a pattern of dead cells in leaves miR399 targets and suppresses the expression of PHO2, of pho2 mutants that have been inoculated with P. cuc- encoding the ubiquitin-conjugating enzyme that mediates umerina spores (Figure 3c), as was also observed in the degradation of Pi transporter proteins (Chiou miR399 OE plants. Finally, the pho2 plants also showed et al., 2006; Fujii et al., 2005; Huang et al., 2013). A loss-of- resistance to C. higginsianum infection as compared with function allele of PHO2 was previously described (Aung wild-type plants (Figure 3d). Taken together, results here et al., 2006; Delhaize & Randall, 1995). This mutant allele presented support the notion that both pho2 and miR399 possesses a single nucleotide mutation that causes prema- OE plants accumulate Pi in leaves and exhibit resistance to ture termination and loss of ubiquitin conjugation activity infection by fungal pathogens with a necrotrophic or hemi- of PHO2 (Figure S2a). pho2 plants resemble miR399 OE biotrophic lifestyle. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 456 Beatriz Val-Torregrosa et al. (a) C. higginsianum Figure 2. Resistance of miR399 OE plants to infec- tion by the hemibiotrophic fungus C. higginsianum. Leaves were locally inoculated with a spore suspen- sion at 4 9 106 spores ml1. Results are from one Fungal Biomass out of three independent experiments performed with three independent miR399 OE lines (lines 1, 4, and 10) and wild-type plants which gave similar results. At least 12 plants per genotype were assayed in each experiment. (a) Disease symptoms at 7 days post-inoculation (dpi) with fungal spores. The diseased leaf area was quantified using ImageJ software. Quantification of C. higginsianum DNA was carried out by qPCR using specific primers for the C. higginsianum Internally transcribed spacer 2 (ITS2) gene at 7 dpi. Values are fungal DNA levels 20 normalized against the Arabidopsis UBIQUITIN21 gene (At5g25760). Comparisons have been made 15 relative to wild-type plants. Histograms show the mean  SEM (t-test, *P ≤ 0.05, **P ≤ 0.01). (b) Try- 10 pan blue staining of C. higginsianum-infected leaves of wild-type and miR399 OE plants at 7 dpi. h, * hyphae. Arrows and arrowheads indicate fungal 5 ** hyphae and dead cells, respectively. Higher magnifi- ** cations of these regions are shown (right panels). 0 Bars represent 100 lm. WT #1 #4 #10 WT miR399 OE (b) WT h h miR399 OE MIR399 expression is upregulated during fungal infection examined at 48 and 72 h post-inoculation (hpi) with P. cuc- and treatment with fungal elicitors umerina spores. An increase in the accumulation of both To gather further support for the involvement of miR399 in precursor and mature miR399 sequences was clearly Arabidopsis immunity, we investigated whether fungal observed in the response of wild-type plants to pathogen infection or treatment with fungal elicitors provokes alter- infection (Figure 4a). When examining the PHO2 expres- ations in MIR399 expression in wild-type plants. The accu- sion behavior in response to infection, an opposite profile mulation of miR399f precursor transcripts was examined was observed between MIR399f and PHO2 at the earliest by reverse transcription qPCR (RT-qPCR), while mature time of infection here assayed (48 hpi with P. cucumerina miR399 abundance was determined by stem-loop RT- spores). At 72 hpi, however, the accumulation of PHO2 qPCR. Differences among mature miR399 sequences are transcripts in fungal-infected wild-type plants was similar found at their 30 terminal region (miRBase, https://www. to that in mock-inoculated plants (Figure 4a). mirbase.org/). This fact allowed us to design miR399f- In PTI, the activation of defense mechanisms relies on specific primers for stem-loop RT-qPCR analysis. The PCR- the detection of PAMPs (commonly referred to as elicitors). amplified products were further confirmed through DNA Accordingly, we investigated whether miR399 accumula- sequencing. Fungal-responsiveness of MIR399f was tion is affected by treatment with a crude preparation of Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 457 Figure 3. Resistance to infection by fungal patho- (a) P. cucumerina gens in pho2 plants. Three-week-old mutant plants were inoculated with fungal P. cucumerina spores. Three independent experiments were carried out with similar results with at least 12 plants per geno- type. (a) Disease phenotype of wild-type and pho2 plants upon inoculation with P. cucumerina spores (5 9 105 spores ml1). Pictures were taken at 7 days post-inoculation (dpi). (b) Survival ratio of wild-type and pho2 plants at 7 dpi (left panel). Quantification of P. cucumerina DNA was carried out using specific primers for P. cucumerina b- tubulin at 7 dpi (right panel). Values are fungal DNA levels normalized against the Arabidopsis UBI- WT pho2 QUITIN21 gene (At5g25760). Data are mean  SEM (n = 7) (t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (c) Trypan blue staining of P. cucumerina-infected leaves and visualization of cell death and fungal (b) 100 ** growth. h, hyphae. Arrows and arrowheads indicate fungal hyphae and dead cells, respectively. Bars represent 200 lm. (d) Disease phenotype of wild- Survival (%) type and pho2 plants at 8 dpi with C. higginsianum spores (4 9 106 spores ml1). 50 0 WT pho2 (c) WT h WT h W T pho2 (d) C. higginsianum 150 Fungal Biomass 100 WT 50 pho2 *** 0 WT pho2 Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 458 Beatriz Val-Torregrosa et al. (a) 0.12 Pre-miR399 0.04 miR399 0.4 Pho2 b Relative expression 0.03 b 0.3 0.08 0.02 0.2 0.04 ab a 0.01 0.1 ab a a a 0.00 0.00 0.0 48h 72h 48h 72h 48h 72h (b) 0.20 Pre-miR399 5 miR399 0.010 Pho2 b b 4 0.008 0.15 3 0.006 0.10 2 0.004 0.05 1 0.002 a a a a a a 0.00 0 0.000 90min 120min 90min 120min 90min 120min Figure 4. Acumulation of precursor (pre-miR399), mature (miR399), and PHO2 transcripts in wild-type plants in response to inoculation with P. cucumerina spores or treatment with P. cucumerina elicitors. The accumulation of pre-miR399f and mature miR399f sequences was determined by RT-qPCR and stem-loop RT-qPCR, respectively, at the indicated times after inoculation with fungal spores (a) or elicitor treatment (b). At 48 and 72 hpi with P. cucumerina spores, pre- miR399 levels increased 4.5- and 6.0-fold, respectively. At the same time points, miR399f levels increased 2.0- and 9.0-fold. Elicitor treatment elevated the level of pre-miR399f accumulation 5.0- and 267-fold at 90 and 120 min of treatment, respectively. MiR399f levels increased 20- and 147-fold in response to elicitor treatment (90 and 120 min, respectively). Four biological replicates and three technical replicates per time point were assayed. Statistically significant differences were determined by ANOVA followed by Tukey’s HSD test, where different letters represent statistically significant differences. elicitors obtained by autoclaving and sonicating P. cuc- of Arabidopsis plants to salt stress (Pegler et al., 2021). In umerina mycelium. Similar to P. cucumerina infection, elic- that study, no anticorrelation between miR399 accumula- itor treatment induced the accumulation of both miR399 tion and PHO2 transcript abundance was reported. precursor and mature sequences (Figure 4b). Furthermore, Whether additional factors are involved in the regulation of an opposite profile was observed between MIR399 and PHO2 during pathogen infection deserves further investi- PHO2 expression at 90 min of elicitor treatment (e.g., gation. upregulation of MIR399f and downregulation of PHO2), but Stimulation of ROS production in Arabidopsis plants this anticorrelation in expression patterns was not containing increased levels of Pi observed at 120 min of elicitor treatment. The observation that pathogen infection and treatment with fungal elicitors One of the hallmarks of host–pathogen interactions is the are accompanied by upregulation of MIR399 expression overproduction of ROS as a plant defense mechanism, the suggests that miR399 might function in PTI. Taking into so-called oxidative burst. ROS include various forms of account that miR399 has been shown to negatively regu- reactive molecules, such as superoxide radicals (O2.), late PHO2 via the canonical mechanism of cleavage of hydroxyl radicals (OH.), and hydrogen peroxide (H2O2). Of PHO2 transcripts, results here presented point to the exis- these, H2O2 might act as both signaling molecule for the tence of regulatory mechanisms for the control of PHO2 activation of plant immune responses and antimicrobial expression other than miR399-guided cleavage of PHO2 agent (Torres et al., 2006). Respiratory Burst Oxidase transcripts during pathogen infection or treatment with Homolog D (RBOHD), a member of the Arabidopsis Nicoti- fungal elicitor (as inferred from results obtained at 72 hpi namide Adenine Dinucleotide Phosphate (NADPH) oxidase with P. cucumerina spores or 120 min of elicitor treat- gene family, has been shown to be responsible for ROS ment). Supporting this possibility, it was recently reported production after pathogen infection (Kadota et al., 2015; that PHO2 might be modulated by regulatory mechanisms Torres et al., 2002). Furthermore, ROS may promote cell independent of miR399-directed regulation in the response death and limit pathogen spread. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 459 Knowing that miR399 OE and pho2 plants exhibit a pat- exhibited upregulation of RBOHD in the absence of patho- tern of cell death upon pathogen infection, we examined gen infection compared with wild-type plants (Figure 5b, ROS accumulation in miR399 OE and pho2 plants, both in black bars). Plectosphaerella cucumerina infection further the presence and in the absence of pathogen infection. His- induced RBOHD expression in all the genotypes (wild-type, tochemical detection of H2O2 was carried out using the flu- miR399 OE, and pho2 plants), but its expression reached orescent probe 2’,7’-dichlorofluorescein diacetate higher levels in miR399 OE and pho2 plants than in wild- (H2DCFDA). H2DCFDA was previously shown to detect dif- type plants (Figure 5b, gray bars). Then, ROS accumulation ferent forms of ROS, mainly H2O2 but also hydroxyl radi- in miR399 OE and pho2 plants can be explained, at least cals and superoxide anions (Fichman et al., 2019). partially, by an increased RBOHD expression. However, Compared with wild-type plants, a higher level of ROS other factors causing an increase in ROS accumulation accumulation could be observed in leaves of miR399 OE cannot be discarded. and pho2 plants compared to wild-type plants in the The observation that both miR399 OE and pho2 plants absence of pathogen infection (Figure 5a, upper panels). accumulated Pi and ROS, these plants also exhibiting Plectosphaerella cucumerina infection further increased enhanced disease resistance (Figures 1 and 2), prompted ROS levels in miR399 OE and pho2 plants (Figure 5a, lower us to investigate whether ROS production and disease panels). Discrete regions accumulating ROS, most proba- resistance are affected by Pi supply in Arabidopsis. To bly, correspond to infection sites. Similar results were address this question, wild-type plants were grown observed by 3,3’-diaminobenzidine (DAB) staining of P. in vitro on media at different Pi concentrations (0.05, 0.1, cucumerina-infected leaves (Figure S3). Moreover, and 0.25 mM Pi, hereinafter referred to as P0.05, P0.1, and H2DCFDA staining revealed a higher level of ROS accumu- P0.25 plants). As expected, measurement of Pi content lation in high Pi plants (e.g., miR399 OE and pho2 plants) confirmed that increasing Pi supply to wild-type plants that have been treated with P. cucumerina elicitors com- results in higher leaf Pi content (Figure 5c). Most impor- pared with elicitor-treated wild-type plants (Figure S4). tantly, increasing Pi supply was accompanied by an Additionally, we examined ROS generated in miR399 increase in ROS accumulation as revealed by H2DCFDA OE, pho2, and wild-type plants in response to treatment staining of leaves from Pi-treated plants (Figure 5d). Sim- with P. cucumerina elicitors using the luminol method. ilarly, luminol assays demonstrated that treatment with This study confirmed a higher production of ROS after elic- Pi fosters ROS production in wild-type plants, with ROS itor treatment in leaves of miR399 OE and pho2 plants production correlating well with Pi concentration (Fig- compared to that in wild-type plants (Figure S5a). Collec- ure S5b). Upon pathogen inoculation, high Pi plants con- tively, these findings support ROS accumulation in leaves sistently displayed enhanced resistance to infection by of miR399 OE and pho2 plants in the absence of pathogen either P. cucumerina or C. higginsianum (Figure 5e,f, infection. During infection with P. cucumerina or treatment respectively). with elicitors obtained from this fungus, miR399 OE and Pi-induced resistance to pathogen infection in Arabidopsis pho2 plants produced higher levels of ROS than wild-type is associated with modulation of the SA- and JA- plants. dependent defense pathways In concordance with results obtained by histochemical detection of ROS and measurement of ROS production As previously mentioned, SA and JA play a critical role in using the luminol assay, the miR399 OE and pho2 plants the transcriptional reprogramming of Arabidopsis plants in Figure 5. Enhanced accumulation of ROS and disease resistance in Arabidopsis plants that overaccumulate Pi. (a) In situ histochemical detection of ROS in leaves of miR399 OE and pho2 plants. Plants were spray-inoculated with P. cucumerina spores (5 9 105 spores ml1) or mock-inoculated. Visualization of H2O2 accumulation was carried out using the fluorescent probe H2DCFDA at 2 days post-inoculation (dpi). Bars represent 200 lm. (b) Expression of RBOHD in mock- inoculated and P. cucumerina-inoculated plants at 24 hpi (black and gray bars, respectively). The expression values were normalized to the Arabidopsis b- tubulin2 gene (At5g62690). Three biological replicates (with three plants per replicate) were examined. Different letters represent statistically significant differ- ences (ANOVA followed by Tukey’s HSD test; P < 0.05). (c) Free Pi content in plants that have been grown under different conditions of Pi supply. Plants were grown on agar plates for 1 week and transferred to fresh agar plates with medium containing different concentrations of Pi (0.05, 0.1, or 0.25 mM). Plants were allowed to continue growth for one more week and then inoculated with fungal spores. Pi content was determined at the time of inoculation with fungal spores. (d) ROS accumulation in wild-type Arabidopsis (Col-0) plants that have been grown under different Pi supply conditions, that is, 0.05, 0.1, 0.25, or 2 mM Pi (P0.05, P0.1, P0.25, and P2, respectively). ROS were detected using H2DCFDA. Representative images are shown. Bars correspond to 1 mm. (e) Resistance to infection by P. cucumerina in Pi-treated Arabidopsis plants. Appearance of plants at 7 dpi) with P. cucumerina spores (4 9 106 spores ml1; left panel). Representative results from one of three independent infection experiments that gave similar results are shown. Right panel, fungal biomass determined at 3 dpi by qPCR anal- ysis using specific primers for P. cucumerina b-tubulin and normalized to the Arabidopsis UBIQUITIN21 gene (At5g25760). (f) Resistance of Pi-treated Arabidop- sis plants to infection by C. higginsianum. Disease symptoms of Arabidopsis plants at 12 dpi with C. higginsianum spores (5 9 105 spores ml1). Right panel, fungal biomass at 7 dpi as determined by qPCR analysis using specific primers for C. higginsianum Internally transcribed spacer 2 (ITS2). Means of three biolog- ical replicates, each one from a pool of at least three plants, are shown in (e) and (f) (right panels; t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 460 Beatriz Val-Torregrosa et al. (a) pho2 (b) WT OE #1 OE #10 Relative expression Non-infected + P. cucumerina (c) (d) P0.05 P0.1 P0.25 P2 (e) P. cucumerina P0.05 P0.1 P0.25 Fungal Biomass (f) C. higginsianum P0.1 P0.25 32 ** P0.05 24 16 ** 8 0 P0.05 P0.1 P0.25 Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 461 response to pathogen infection (Pieterse et al., 2012). A Further, we measured the levels of the phytohormones pathogen-induced accumulation of ROS is also required SA and JA in miR399 OE and pho2 plants. Compared with for the induction of SA-dependent defenses, indicating that wild-type plants, miR399 OE and pho2 plants accumulated ROS and SA are intertwined in a complex regulatory net- significantly higher levels of SA under normal growth con- work (Herrera-Vasquez et al., 2015; Wang et al., 2014). To ditions (e.g., in the absence of pathogen infection) (Fig- get deeper insights into the mechanisms underlying dis- ure 7a, upper left panel, black bars). This observation is ease resistance in miR399 OE and pho2 plants, we investi- consistent with the expression pattern of SA-responsive gated the expression of defense genes linked to the SA defense genes found in miR399 OE and pho2 plants (see and JA pathways in these plants. The marker genes of the Figure 6). Upon pathogen infection, however, there were no SA-mediated defense response here examined were: significant differences in SA levels among wild-type, Pathogenesis-Related 1 (PR1), Non-expressor of miR399 OE, and pho2 plants (Figure 7a, upper left panel, Pathogenesis-Related genes 1 (NPR1), and Phytoalexin gray bars). We also noticed that SA glucoside (SAG; the Deficient4 (PAD4) (Jirage et al., 1999). We found that in the storage form of SA) accumulated at substantially higher absence of pathogen infection, PR1, NPR1, and PAD4 levels in the fungal-infected miR399 OE and pho2 plants expression was upregulated in both miR399 OE and pho2 compared with the fungal-infected wild-type plants (Fig- plants compared with wild-type plants (Figure 6a, black ure 7a, upper right panel). These observations points to a bars). PR1, NPR1, and PAD4 expression further increased strict control of the SA level in miR399 OE and pho2 plants. in response to P. cucumerina infection in miR399 OE and In the absence of pathogen infection, miR399 OE and pho2 plants, but not in wild-type plants (Figure 6a, gray pho2 plants accumulated more JA than wild-type plants bars). (Figure 7a, lower left panel, black bars). JA content further Regarding the JA pathway, two branches are documented increased during pathogen infection in wild-type and in Arabidopsis: the MYC2 branch, which is regulated by miR399 OE plants (Figure 7a, lower left panel, gray bars). AtMYC2 (a basic helix–loop–helix–leucine zipper transcription The level of 12-oxophytodienoic acid (OPDA), a JA precur- factor), and the ERF branch, which is regulated by AtERF1 (a sor, in miR399 OE and pho2 plants did not differ signifi- member of the APETALA/ERF transcription factor family) cantly from that in wild-type plants (Figure 7a). (Lorenzo et al., 2004). Plant Defensin 1.2 (PDF1.2) is com- Additionally, we investigated whether Pi treatment mod- monly used as marker of the ERF branch, whereas the MYC ulates SA and JA signaling pathways. Increasing Pi supply branch is marked by the induction of Vegetative Storage Pro- results in a gradual increase in SA and JA levels (as well tein 2 (VSP2) (Lorenzo et al., 2004; Pieterse et al., 2012; as SAG and OPDA levels) (Figure 7b). These results are in Wasternack & Hause, 2013; Zhang et al., 2017). The ERF concordance with the upregulation of PR1, NPR1, and branch and the MYC branch of the JA signaling pathway PDF1.2 in response to Pi treatment (Figure S6). have been reported to repress each other (Aerts et al., 2021; Collectively, these results support the notion that in the Lorenzo et al., 2004; Wasternack & Hause, 2013). absence of pathogen infection, miR399 overexpression, Compared with wild-type plants, the miR399 OE and loss-of-function of PHO2, and Pi treatment result in higher pho2 plants showed higher expression of transcription fac- levels of SA and JA and subsequent upregulation of SA- tor and defense marker genes associated to the ERF1/ and JA-dependent defense gene expression. A Pi-mediated PDF1.2 and MYC2/VSP2 branches of the JA signaling path- modulation of the SA and JA signaling pathways may con- way in the absence of pathogen infection (Figure 6b,c, tribute to the phenotype of disease resistance that is black bars). Pathogen infection further induced ERF1/PDF1 observed in Arabidopsis plants overaccumulating Pi, expression in wild-type, miR399 OE, and pho2 plants (Fig- namely miR399 OE, pho2, and wild-type plants grown ure 6b, gray bars). Of note, while MYC2/VSP2 expression under high Pi supply. was not significantly affected by pathogen infection in DISCUSSION wild-type plants, their expression was found to be repressed in miR399 OE plants. Pathogen infection down- Nutrients play crucial roles in normal plant growth and regulated VSP2 expression in pho2 plants, but not in wild- development, and nutrient imbalance might also have a type plants (Figure 6c, gray bars). Together, these findings substantial impact on the predisposition of plants to resist support the notion that resistance to pathogen infection in pathogen attack. Depending on the identity of the interact- plants accumulating Pi (i.e., miR399 OE and pho2 plants) is ing partners, nutritional imbalances caused by either nutri- associated with a higher expression of SA- and JA- ent excess or deficiency may determine the outcome of the regulated genes under non-infection conditions. A interaction, resistance or susceptibility (Veresoglou pathogen-induced superactivation of genes involved in the et al., 2013). On the other hand, although miRNA-mediated SA pathway and the ERF1 branch of the JA pathway occurs regulation of gene expression in processes involved in in these plants, while pathogen infection represses the either nutrient stress or immune responses is well docu- MYC2 branch of the JA signaling pathway. mented, less effort has been made to investigate miRNA Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 462 Beatriz Val-Torregrosa et al. (a) 0.10 NPR1 0.5 PAD4 Relative expression 0.4 0.08 d d cd 0.3 0.05 bcd bc ab 0.2 c 0.03 b b a 0.1 a a 0.00 0.0 WT #1 #10 WT #1 #10 miR399 OE miR399 OE 5 PR1 0.16 NPR1 4 c c 0.12 3 b b b 0.08 2 a a 0.04 1 a 0 0.00 WT pho2 WT pho2 (b) (c) Relative expression Relative expression Relative expression Relative expression Figure 6. Expression of defense genes in miR399 OE and pho2 plants. Transcript levels were determined by RT-qPCR in mock-inoculated or P. cucumerina-inoc- ulated plants at 24 h post-inoculation (black and gray bars, respectively). The expression values were normalized to the Arabidopsis b-tubulin2 gene (At5g62690). (a) Expression of genes involved in the SA pathway (PR1, NPR1, PAD4). (b) Expression of genes in the ERF branch of the JA pathway (ERF1, PDF1.2). (c) Expression of genes in the MYC branch of the JA pathway (MYC2, VSP2). Three independent experiments (with 12 plants per genotype) were examined, with similar results. Bars represent mean  SEM. Different letters represent statistically significant differences (ANOVA followed by Tukey’s HSD test; P < 0.05). function in plant immunity under nutrient stress condi- Several pieces of evidence support this conclusion. On the tions, in particular Pi stress. one hand, we show that Pi accumulation caused by In this study, we provide evidence that increasing Pi con- miR399 overexpression, loss-of-function of PHO2, or treat- tent in Arabidopsis results in enhanced disease resistance. ment with Pi confers resistance to infection by fungal Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 463 Figure 7. Levels of SA, SAG, JA, and OPDA in (a) 15 leaves of Arabidopsis plants that overaccumulate Pi (miR399 OE, pho2, and wild-type plants). (a) Hor- mone levels were measured in mock-inoculated and P. cucumerina-inoculated miR399 and pho2 10 plants at 48 hpi. (b) Hormone levels in wild-type plants that have been grown at the indicated Pi sup- ply conditions (0.05, 0.1, and 0.25 mM Pi). Plants were grown as indicated in Figure 5. Three inde- pendent experiments (with 12 plants per genotype) were examined, with similar results. Bars represent mean  SEM. Different letters represent statistically significant differences (ANOVA followed by Tukey’s HSD test; P < 0.05). ng/g dw (x10-3) (b) 10 pathogens with necrotrophic (P. cucumerina) and hemibio- pathogen infection). Additionally, we show that P. cuc- trophic (C. higginsianum) lifestyles. On the other hand, umerina infection is accompanied by an increase in resistance in Arabidopsis plants overaccumulating Pi corre- miR399 accumulation. Not only pathogen infection, but lated well with upregulation of defense-related genes also treatment with fungal elicitors results in higher levels under normal growth conditions (i.e., in the absence of of miR399. The observed elicitor-responsiveness of MIR399 Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 464 Beatriz Val-Torregrosa et al. expression might be indicative that miR399 functions in forward loop between SA and ROS production (e.g., H2O2) PTI. Altogether, these findings support the notion that has been reported in which ROS are involved both miR399 overexpression has a positive impact on resistance upstream and downstream of SA in the plant defense to pathogen infection in Arabidopsis and reinforce the response to pathogen infection (Herrera-Vasquez notion that miR399 plays a dual role in plants by control- et al., 2015). We hypothesize that Pi content and possibly ling Pi homeostasis and disease resistance. also ROS accumulation might influence defense hormone Another finding of this study is that, under non-infection signaling. conditions, overaccumulation of Pi in Arabidopsis leaves is Interestingly, a different regulation in the two branches accompanied by production of ROS, their levels increasing of the JA pathway was observed in high Pi plants during significantly during pathogen infection. Hence, ROS accu- pathogen infection. Whereas ERF1 and PDF1.2 expression mulation in miR399 OE and pho2 plants might be responsi- is further increased during infection in miR399 OE and ble for the pattern of cell death observed in these plants pho2 plants (infected versus non-infected plants), VSP2 during pathogen infection, a response that is reminiscent expression diminished in miR399 OE and pho2 plants (in- of the pathogen-induced HR. In contrast, the fungal- fected versus non-infected plants). We envisage that this infected wild-type plants did not exhibit cell death and differential regulation might be due to still unknown fac- showed extensive fungal growth. Additionally, elevated tors that cooperate in an antagonistic manner in the regu- levels of ROS might contribute to the activation of immune lation of PDF1.2 and VSP2 expression during infection with responses in high Pi plants leading to a phenotype of dis- the fungal pathogen P. cucumerina. In line with this, a neg- ease resistance. From this point of view, it might be inter- ative correlation between the ERF1 and MYC2 branches esting to determine whether Pi-induced ROS accumulation has been previously described in the Arabidopsis response can be generalized to other plant species. to different attackers such as pathogens and phytophagous At the time of inoculation with fungal spores (3-week-old insects (Lorenzo et al., 2004; Pieterse et al., 2012; Waster- plants), miR399 OE and pho2 plants accumulated 3–4 times nack & Hause, 2013; Zhang et al., 2017). Here, necrotrophic more Pi than wild-type plants. By this developmental pathogens preferentially activate the ERF branch, while the stage, plant growth is not compromised in plants accumu- MYC2 branch is activated by insect herbivory and wound- lating this level of Pi. It is tempting to hypothesize that this ing. A specialization in the host plant for modulation of increase in Pi content and/or ROS levels is perceived by each branch of the JA signaling pathway might exist. It will the host plant as a stressful situation and that the plant be of interest to explore whether Pi accumulation has an responds to these signals with the induction of defense effect on plant–insect interactions in Arabidopsis. gene expression. An increase in Pi content might eventu- Clearly, interactions between defense-related hormone ally increase intracellular and extracellular Pi levels, pathways provide the plant with a powerful regulatory increasing ATP levels. Here, it should be mentioned that potential to control defense responses (Aerts et al., 2021; ATP has been shown to function as a DAMP signal after Zheng et al., 2012). However, the type of induced response release into the extracellular space upon cellular damage that is effective for disease resistance appears to vary and that extracellular ATP enhances plant defense against depending on the host plant and pathogen identity. pathogens through the activation of JA signaling (Tanaka Although there are exceptions, pathogens with a bio- et al., 2014; Tripathi et al., 2018). Further studies are trophic or hemibiotrophic lifestyle (such as P. syringae) are needed to establish whether ATP levels are altered in high generally more sensitive to SA-dependent responses, Pi plants. whereas necrotrophic pathogens are commonly deterred Gene expression analysis revealed regulation of the SA by JA/ET-dependent defenses (Glazebrook, 2005). The and JA defense signaling pathways in Arabidopsis plants observation that high Pi plants (e.g., miR399 OE, pho2, and overaccumulating Pi in leaves. Thus, high Pi plants plants grown under high Pi supply conditions) show (miR399 OE and pho2 plants) showed activation of SA- enhanced resistance to necrotrophic (P. cucumerina) and regulated and JA-regulated genes under non-infection con- hemibiotrophic (C. higginsianum) fungal pathogens makes ditions. SA and JA were reported to play a positive role in it unlikely that the pathogen lifestyle determines disease the regulation of resistance to P. cucumerina infection in resistance in high Pi plants. Arabidopsis (Berrocal-Lobo et al., 2002; Sanchez-Vallet Based on the results obtained in this study, a model is et al., 2012). Previous studies also revealed that PDF1.2 proposed to describe possible mechanisms underlying dis- induction was associated to resistance to infection by ease resistance in high Pi Arabidopsis plants (Figure 8). necrotrophic fungi, including P. cucumerina, in Arabidop- According to our model, miR399 overexpression and loss- sis (Berrocal-Lobo et al., 2002; Thomma et al., 1998). Con- of-function of PHO2, as well as growing plants under high sistent with the upregulation of genes involved in the SA Pi supply, increases Pi accumulation, ROS production, and and JA signaling pathways, SA and JA accumulated in SA and JA accumulation. A higher expression of SA- and non-infected high Pi plants. In other studies, a feed- JA-dependent defense responses in Arabidopsis plants Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 465 High-Pi Supply miR399 overexpression / pho2 transgenic expression of a phytoplasma effector (SAP11) in Arabidopsis was found to trigger PSRs that are mainly dependent on PHR1 (Lu et al., 2014). The SAP11 transgenic Pi plants overaccumulated Pi in leaves and were more sus- ceptible to P. syringae pv. tomato DC3000 infection (Lu et al., 2014). The PSR system also appears to control root ? colonization by the endophytic fungus Colletotrichum RBOHD ROS SA JA tofieldiae in Arabidopsis (Frerigmann et al., 2021; Hiruma No infecon et al., 2016). Very recently, Dindas et al. (2022) described a NPR1 ERF1 PAD4 PDF1.2 negative regulation of Pi transport by immune signaling in PR1 Arabidopsis. In citrus plants, Pi content was associated to MYC2 symptomology in Huanglongbing (HLB) disease, where Pi VSP2 deficiency favors development of HLB symptoms (Zhao et al., 2013). In plant–insect interactions, Pi deficiency was found to induce the JA signaling pathway to enhance resistance to insect herbivory in a process that is partially RBOHD ROS SA JA under the control of PHR1 (Khan et al., 2016). Collectively, results here presented together with those found in the lit- Infecon NPR1 ERF1 erature in other pathosystems support the existence of PAD4 PDF1.2 connections between Pi homeostasis and immune signal- PR1 ing. These mechanisms should operate in a coordinated VSP2 manner to properly balance nutrient responses and plant immunity. Results from our study also raise intriguing questions Disease Resistance about the impact of Pi content on disease resistance in dif- ferent pathosystems. In the present study we show that an Figure 8. Proposed model to explain how Pi content and defense responses increase in Pi content positively regulates Arabidopsis are integrated for modulation of resistance to infection by fungal pathogens in Arabidopsis. Treatment with high Pi, MIR399 overexpression, and loss-of- immune responses, which, in turn, enhances resistance to function of PHO2 would trigger Pi accumulation, which, in turn, would infection by fungal pathogens. Contrarily, in rice plants, a increase ROS and hormone (SA and JA) levels in Arabidopsis leaves for the higher Pi content caused by miR399 overexpression or induction of genes involved in the SA and JA signaling pathways. Upon pathogen infection, the expression of genes involved in the SA pathway high Pi fertilization was found to negatively regulate and the ERF1/PDF1.2 branch of the JA pathway would be further induced, defense gene expression, thus increasing susceptibility to while genes in the MYC2/VSP2 branch of the JA pathway are repressed. infection by the blast fungus M. oryzae (Campos-Soriano The interplay between Pi, ROS, and hormones would allow the plant to mount an effective immune response. Although crosstalk between ROS and et al., 2020). In other studies, Pi deficiency was found to hormonal pathways has been described (Herrera-Vasquez et al., 2015; Xia enhance resistance to Verticillium dahliae in cotton et al., 2015), further investigation is needed to elucidate (i) the exact mecha- (Gossypium hirsutum) (Luo et al., 2021) and insect her- nisms by which Pi content modulates ROS production and hormone con- tent and (ii) how these signaling pathways communicate with each other in bivory in Arabidopsis (Khan et al., 2016). Together, our the coordinated regulation of Pi homeostasis and immune responses. results and those previously reported support the notion that Pi content might positively or negatively regulate dis- ease resistance depending on the interacting partners. It is accumulating Pi would allow the plant to mount a success- tempting to speculate that different plants might have ful defense response upon encountering a pathogen. evolved diverse mechanisms to adapt to Pi alterations The existence of links between components of the phos- which, in turn, would determine a different effect of Pi in phate starvation response (PSR) and disease resistance the regulation of immune responses. It is likely that inte- has been previously described (Chan et al., 2021). For gration of Pi stress (either deficiency or excess) and instance, enhanced resistance to infection by the bacterial immune responses might vary depending on the host pathogen P. syringae pv. tomato DC3000 and the oomycete plant and the type of pathogen, the outcome of the interac- pathogen Hyaloperonospora arabidopsidis was reported in tion being also dependent on the role of the defense hor- phr1 mutant plants, PHR1 being the master transcriptional mones SA and JA in that particular interaction. regulator of the Arabidopsis PSR (Castrillo et al., 2017). Alternatively, Pi might affect growth and/or pathogenicity This study also led the authors to propose that PHR1 might of a fungal pathogen, either by creating a less favorable fine-tune JA responses under Pi starvation in specific bio- environment for pathogen growth or by reducing the pro- logical contexts, rather than being a regulator of the JA duction of pathogen virulence factors. Clearly, these signaling pathway (Castrillo et al., 2017). In other studies, aspects deserve further investigation. Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 466 Beatriz Val-Torregrosa et al. Finally, it is worth mentioning that one of the major ref. CS8508). A point mutation in the sixth exon (from G2539 to A, challenges plants face is defending against pathogen infec- relative to the translational start codon) causes an early termina- tion at the beginning of the UBC domain, thus resulting in the loss tion under continuous changes in nutrient availability, par- of the ubiquitin-conjugating activity of PHO2 in the pho2 mutant ticularly Pi availability. Results here presented set the basis (Aung et al., 2006). for further research to elucidate the exact mechanisms by which Pi signaling and pathogen-induced signaling interact Plant tissue staining with each other in plants. Further studies, however, should For trypan blue staining, leaves were fixed by vacuum infiltration be conducted on a case-by-case basis in different plant– for 1 h in ethanol:formaldehyde:acetic acid (80:3.5:5 v/v), stained pathogen interactions. A better understanding of these with lactophenol blue solution for 1 h, and then washed with chlo- mechanisms will allow the development of novel strategies ral hydrate for 15 min. Leaves were placed on glass slides with glycerol and observed using a Leica DM6 microscope under bright to improve disease resistance in plants. field conditions. For H2DCFDA staining, Arabidopsis leaves were placed on a EXPERIMENTAL PROCEDURES solution of H2DCFDA (at a concentration of 10 lM), vacuum infil- trated during 5 min, and then maintained in darkness for 10 min. Plant material and infection assays Two washes with distillated water were performed. Photographs Arabidopsis thaliana (ecotype Columbia-0 [Col-0]) plants were were taken on a Leica DM6 microscope to visualize green fluores- grown in a mixture of soil:perlite:vermiculite (2:1:1) and modified cence. DAB staining was also used to examine H2O2 levels. For Hoagland half strength medium, under neutral photoperiod (12 h this, Arabidopsis plants were immersed in a DAB solution light /12 h dark), at a relative humidity of 60% and a temperature (1 mg ml1) for 30 min with vacuum treatment, maintained during of 22°C  2°C. The fungus P. cucumerina was grown on Potato 4 h in darkness under agitation, washed with 95% ethanol for Dextrose Agar plates with chloramphenicol (34 lg ml1). Col- 30 min at 75°C, and observed using a Leica DM6 microscope letotrichum higginsianum was grown on Oatmeal agar plates in under bright field illumination. darkness. Fungal spores were collected by adding sterile water to ROS production in response to treatment with P. cucumerina the surface of the mycelium and adjusted to the desired final con- elicitors (300 lg ml1) was measured using the luminol method centration using a Bu € rker counting chamber. described by Smith and Heese (2014). Measurements were carried For infection experiments in soil-grown plants, 3-week-old out on a CentroXS3 LB 960 Microplate reader (Berthold Technolo- plants were spray-inoculated with a spore suspension of P. cuc- gies, Bad Wildbad, Germany). umerina (5 9 105 spores/ml) or mock-inoculated. Plectosphaerella Generation of transgenic Arabidopsis plants cucumerina-inoculated and mock-inoculated plants were main- tained under high humidity and plant survival was assessed at For MIR399f overexpression, the miR399 precursor sequence was 7 dpi. For infection with C. higginsianum, the fungal spores were cloned under the control of the Cauliflower mosaic virus (CMV) locally inoculated (4 9 106 spores ml1; 10 ll per leaf and five 35S promoter in the PMDC32 plasmid (Chiou et al., 2006). The leaves per plant). The lesion area of C. higginsianum-infected plant expression vector was transferred to Agrobacterium tumefa- leaves was measured with ImageJ software (National Institute of ciens strain GV301. Arabidopsis Col-0 plants were transformed Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/) at 7 dpi. using the floral dip method. Homozygosis was achieved by antibi- Three independent experiments were performed with at least 12 otic selection. Segregation analysis confirmed transgene inheri- plants per genotype in each experiment. For in vitro experiments, tance in successive generations of transgenic lines. 2-week-old Arabidopsis plants were spray-inoculated with P. cuc- umerina (4 9 106 spores ml1) or locally inoculated with C. higgin- Measurements of Pi content and chlorophyll content sianum (5 9 105 spores ml1). Fungal biomass was determined by quantitative PCR (qPCR) using specific primers for the correspond- The Pi content of Arabidopsis plants was determined as previ- ing fungus; the Arabidopsis UBIQUITIN21 (At5g25760) gene was ously described (Versaw & Harrison, 2002). Chlorophylls were used as the internal control (Soto-Suarez et al., 2017). PCR primers extracted and quantified spectrophotometrically (SpectraMax M3, are listed in Table S1. Statistically significant differences were Molecular Devices, San Jose, CA, USA) as previously described determined by the t-test. (Lichtenthaler & Buschmann, 2001). Elicitor treatments were performed by spraying 3-week-old Gene expression analyses plants with an elicitor extract obtained from the fungus P. cuc- umerina (300 lg ml1) as previously described (Casacuberta Total RNA was extracted using TRIzol reagent (Invitrogen, Wal- et al., 1992). Three independent experiments were performed with tham, MA, USA). First-strand cDNA was synthesized from DNase- at least 12 plants per genotype in each experiment. treated total RNA (1 lg) with reverse transcriptase and oligo-dT For Pi treatment experiments, plants were grown in vitro on (High Capacity cDNA reverse transcription kit, Applied Biosys- meshes placed on agar plates with modified Hoagland half tems, Waltham, MA, USA). RT-qPCR was performed in optical 96- strength medium containing 0.25 mM KH2PO4 for 1 week. Seed- well plates using SYBRâ green in a LightCycler 480 (Roche, Basel, lings were then transferred to fresh agar medium at the desired Switzerland). Primers were designed using Primer-Blast (https:// concentration of Pi (0.05, 0.1, 0.25, or 2 mM Pi). The plants were www.ncbi.nlm.nih.gov/tools/primer-blast/). The b-tubulin2 gene allowed to continue growing for one more week under each Pi (At5g05620) was used to normalize the transcript levels in each regime. The in vitro-grown plants were then inoculated with a sample. Primers used for RT-qPCR and stem-loop RT-qPCR are spore suspension of P. cucumerina or C. higginsianum as above. listed in Table S1. Accumulation of mature miR399f was deter- The pho2 mutant, previously named ubc24 (UBIQUITIN- mined by stem-loop RT-qPCR (Varkonyi-Gasic et al., 2007). DNA CONJUGATING ENZYME 24; At2g33770; Col-0 background) was sequencing confirmed the specificity of the PCR-amplified prod- obtained from the Arabidopsis Biological Resource Center (ABRC, ucts. Two-way analysis of variance (ANOVA) followed by Tukey’s Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452–469 Phosphate-induced disease resistance in Arabidopsis 467 honestly significant difference (HSD) test was used to analyze RT- CONFLICT OF INTEREST qPCR data. The authors declare that they do not have competing inter- Hormone determination ests. The rosettes of 3-week-old wild-type (Col-0), miR399 OE, and pho2 DATA AVAILABILITY STATEMENT plants were analyzed by LC–MS for SA, SAG, JA, and OPDA con- tent as previously described (Sanchez-Bel et al., 2018). Briefly, All relevant data can be found within the published article 30 mg of freeze-dried material was extracted with MeOH:H2O and its supporting material. (30:70) containing 0.01% of HCOOH and a mixture of 10 lg L1 of the internal standards SA-d5 and dehydro-JA (Sigma-Aldrich, St. SUPPORTING INFORMATION Louis, MO, USA). Following extraction, samples were centrifuged (25 000 g, 15 min) and filtered through regenerated cellulose fil- Additional Supporting Information may be found in the online ver- ters. An aliquot of 20 ll was injected into a UPLC system (Waters sion of this article. Acquity) interfaced with a Xevo TQ-S Mass Spectrometer (Waters, Milford, MA, USA). Hormones were quantified by contrasting with Figure S1. Phenotypical analysis of miR399 OE plants. an external calibration curve of pure chemical standards of SA, Figure S2. Analysis of pho2 mutant plants under non-infection SAG, JA, and OPDA. Sample separation was performed with an conditions. LC Kinetex C18 analytical column with a particle size of 5 lm, 2.1 Figure S3. ROS accumulation in leaves of high Pi plants (miR399 9 100 mm (Phenomenex, Torrance, CA, USA). Chromatographic OE, pho2 plants) under non-infection conditions and upon infec- and MS conditions were as described in Sanchez-Bel et al. (2018). tion with P. cucumerina as determined by DAB staining. At least six biological replicates were analyzed per genotype and Figure S4. ROS accumulation in leaves of high Pi plants (miR399 condition, each replicate consisting of leaves from at least three OE, pho2 plants) in response to treatment with P. cucumerina elic- independent plants. The plant material was lyophilized prior to itors as determined by H2DCFDA staining. analysis. Two-way ANOVA followed by Tukey’s HSD test was used Figure S5. ROS production in leaves of miR399 OE, pho2, and to analyze data. wild-type plants in response to treatment with P. cucumerina elici- tors as determined using the luminol assay. ROS production in ACCESSION NUMBERS wild-type plants that have been grown under different Pi supply b-Tubulin 2 (At5g62690); PHO2 (At2g33770); PR1 (At2g14610); was also determined using the luminol assay. NPR1 (At1g64280); PAD4 (At3g52430); ERF1 (At3g23240); PDF1.2 Figure S6. Expression of defense-related genes in wild-type Ara- (At2g26020); MYC2 (At1g32640); VSP2 (At5g24770); RBOHD bidopsis plants that have been grown under different Pi supply (At5g47910); Ubiquitin 21 (At5g25760). (0.05, 0.1, or 0.25 mM). ACKNOWLEDGMENTS Table S1. List of oligonucleotides. We thank A. Molina and R.J. O’Connell for providing the P. cuc- REFERENCES umerina and C. higginsianum strains and Glo ria Escola for assis- Aerts, N., Pereira Mendes, M. & Van Wees, S.C.M. (2021) Multiple levels of tance in parts of this work. We acknowledge support of the crosstalk in hormone networks regulating plant defense. The Plant Jour- publication fee by the CSIC Open Access Publication Support Initia- nal, 105, 489–504. tive through its Unit of Information Resources for Research (URICI). Andersen, E., Ali, S., Byamukama, E., Yen, Y. & Nepal, M. (2018) Disease This research was supported by the Ministry of Science, Innovation resistance mechanisms in plants. 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