Received: 25 September 2019 | Revised: 18 December 2019 | Accepted: 10 January 2020 DOI: 10.1111/mpp.12916 ORIGINAL ARTICLE Phosphate excess increases susceptibility to pathogen infection in rice Lidia Campos-Soriano1 | Mireia Bundó1 | Marcel Bach-Pages1 | Su-Fen Chiang2 | Tzyy-Jen Chiou2 | Blanca San Segundo 1,3 1 Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Universitat Abstract Autònoma de Barcelona, Bellaterra Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to (Cerdanyola del Vallés), Barcelona, Spain 2 soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of 3 Consejo Superior de Investigaciones phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas Científicas, Barcelona, Spain adaptive responses to Pi deficiency have been deeply studied, less is known about Correspondence how plants adapt to Pi excess and how Pi excess might affect disease resistance. We Mireia Bundó, Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibil- Campus Universitat Autònoma de Barcelona, ity to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the Bellaterra (Cerdanyola del Vallés), Barcelona, Spain. causal agent of the blast disease, one of the most damaging diseases of cultivated Email: mireia.bundo@cragenomica.es rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in Present address rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen Marcel Bach-Pages, Department of Plant infection, a weaker activation of defence-related genes occurs in rice plants over- Sciences, University of Oxford, Oxford, UK accumulating Pi in leaves, which is in agreement with the phenotype of blast Funding information susceptibility observed in these plants. These data support that Pi, when in excess, M.B.P. was funded by a “la Caixa” scholarship for PhD studies in Spanish compromises defence mechanisms in rice while demonstrating that miR399 func- universities. This research was supported tions as a negative regulator of rice immunity. The two signalling pathways, Pi by the Ministerio de Ciencia, Innovación y Universidades (MCIU)-Agencia Estatal de signalling and defence signalling, must operate in a coordinated manner in controlling Investigación (AEI) y el Fondo Europeo de disease resistance. This information provides a basis to understand the molecular Desarrollo Regional (FEDER) (BIO2015- 67212-R and RTI2018-101275-B-100) and mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect the Academia Sinica, Taiwan that should be considered in management of the rice blast disease. (AS-SS-106-03-3). We also acknowledge support from the CERCA Programme (“Generalitat de Catalunya”) and the “Severo KEYWORDS Ochoa Programme for Centres of Excellence defence response, Magnaporthe oryzae, miR399, Oryza sativa, phosphate, rice, transcriptomics in R&D” 2016-2019 (SEV-2015-0533). 1 | I NTRO D U C TI O N research on plant responses to environmental stress has been con- ducted on plants subjected to an individual type of stress. In natural Plants have evolved multiple mechanisms to perceive and respond habitats, however, plants are simultaneously exposed to combina- to environmental stimuli, including biotic and abiotic stresses. Most tions of stress factors, and cross-talk between responses induced by Lidia Campos-Soriano and Mireia Bundó contributed equally to this work. 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. © 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd Molecular Plant Pathology. 2020;21:555–570.  wileyonlinelibrary.com/journal/mpp | 555 556 | CAMPOS-SORIANO et al. a particular type of stress may result in positive or negative impact developmental processes and adaptation to biotic and abiotic stress over other stress. (Navarro et al., 2006; Chen, 2009; Li et al., 2010; 2016; Shivaprasad Stress resulting from insufficient or excessive supply of nutri- et al., 2012; Campo et al., 2013; Jeong and Green, 2013; Baldrich ents can have an impact on disease resistance. Nutrient stress- and et al., 2015; Baldrich and San Segundo, 2016; Song et al., 2019). pathogen-induced signalling pathways might interact with one an- Evidence also supports a regulation of nutrient homeostasis by other in a synergistic or antagonistic way and this interaction de- certain miRNAs in plants (Liang et al., 2015; Paul et al., 2015; Chien termines resistance or susceptibility to pathogen infection. Along et al., 2017). The best-known examples of miRNAs involved in nutri- with this, different results are found in the literature on the effect ent homeostasis are miR399 and miR827 (phosphate homeostasis), of addition of nutrients on disease severity, in which both increased miR395 (sulphur homeostasis), and miR826 and miR5090 (nitrogen and decreased disease severity have been reported. For instance, homeostasis). whereas nitrogen fertilization increases blast incidence in rice, it re- The implication of miR399 in the phosphate starvation response duces disease severity caused by Verticillium spp. in Solanum species (PSR) of Arabidopsis plants is well documented (Chiou et al., 2006; (Ballini et al., 2013; Veresoglou et al., 2013). Hsieh et al., 2009; Puga et al., 2017; Ham et al., 2018). In brief, A nutrient of great importance for plant growth is phosphorus, Pi deprivation rapidly increases miR399 accumulation, which causes which is absorbed through the roots almost exclusively in the form of degradation of PHOSPHATE2 (PHO2) transcripts encoding a ubiquitin- inorganic phosphate (Pi). Though the overall content of phosphorus in conjugating E2 enzyme responsible for the ubiquitination and pro- soil is generally high, the low bioavailability of phosphate represents a teasome degradation of Pi transporters (Aung et al., 2006; Chiou limiting factor for plant growth in many natural and agricultural eco- et al., 2006; Liu et al., 2012). In this way, accumulation of miR399 systems (Hinsinger et al., 2011). As a consequence, Pi fertilizers are in response to Pi starvation, and down-regulation of PHO2, commonly used in modern farming to maximize crop yields, leading relieves negative post-transcriptional control of Pi transporters for an to a scenario of Pi excess in agricultural ecosystems. Excessive use of increase in Pi uptake and translocation in Arabidopsis plants. While fertilizers and pesticides not only has a negative economic impact, numerous studies have focused on the molecular mechanisms in- but also causes a number of environmental problems due to soil pol- volved in the plant response to Pi starvation, mostly in the model lution and water eutrophication, and raises serious concerns about dicotyledonous plant Arabidopsis, little attention has been paid to food safety and animal health. However, the effect of Pi excess on understand how crop species cope with Pi excess. disease resistance in crop species is poorly explored. Rice is the most widely consumed staple food for a large part During pathogen infection, massive transcriptional repro- of the world's human population. However, rice production is gramming of gene expression occurs for the activation of plant severely threatened by the blast disease caused by the fungal defence responses (Jones and Dangl, 2006; Boller and He, 2009; pathogen Magnaporthe oryzae (Wilson and Talbot, 2009). In rice, Cook et al., 2015). This process is initiated by the recognition of the Pi starvation signal transduction pathway appears to be similar pathogen-associated molecular patterns (PAMPs) by host immune to that of Arabidopsis. OsPHO2 (originally named leaf tip necrosis receptors at the plasma membrane, which triggers the induction 1, LTN1) is the AtPHO2 ortholog in rice (Hu et al., 2011). Despite of defence responses via multiple signalling cascades, so-called the agronomic importance of rice, little information is available PAMP-triggered immunity (PTI) (Bigeard et al, 2015; Couto and regarding the effect of Pi stress, either deficiency or excess, on Zipfel, 2016). Among others, PTI components include the produc- blast disease resistance. tion of reactive oxygen species (ROS), reinforcement of the cell The aim of this study was twofold. First, to investigate whether wall, activation of protein phosphorylation processes, production Pi supply has an effect on blast disease resistance in rice. Second, of antimicrobial compounds, and accumulation of pathogenesis- to assess whether cross-talk between miR399-mediated signalling related (PR) proteins. Certain microbial pathogens are able to pathways and immune signalling pathways exists in rice. We show deliver effector proteins into the host cell that suppress PTI re- that growing rice plants under high Pi supply, as well as miR399 over- sponses, resulting in plant susceptibility. In turn, many plants have expression, increases Pi level in rice leaves and enhances suscep- evolved another layer of immunity through recognition of micro- tibility to M. oryzae infection. Transcriptome analysis revealed that bial effectors (or host proteins modified by effectors) by proteins miR399 overexpression affects the expression of genes involved encoded by resistance (R) genes. This recognition triggers a rapid in protein phosphorylation and transcription factor genes, as well and effective host defence response, so-called effector-triggered as several defence-related genes. On pathogen challenge, a weaker immunity (ETI) (Jones and Dangl, 2006). Whether nutrient stress induction of defence gene expression is observed in miR399 overex- can modulate host defence responses remains elusive, most prob- pressing plants, as well as in wild-type plants that have been grown ably because nutrient stress and disease resistance have been tra- under high Pi fertilization, which correlates well with their enhanced ditionally investigated separately from each other. disease susceptibility phenotype. These data further support a link MicroRNAs (miRNAs) are small noncoding RNAs that mediate between Pi signalling and immune signalling in rice plants. Therefore, post-transcriptional gene silencing through degradation or transla- Pi excess caused by the indiscriminate use of Pi fertilizers in rice tional repression of target transcripts (Llave et al., 2002; Brodersen fields might have unintended consequences in rice production by et al., 2008). They are essential regulators of gene expression in plant facilitating blast infection. CAMPOS-SORIANO et al. | 557 2 | R E S U LT S (Figures 1 and 2). That low-Pi plants perceive and react to the low-Pi treatment used in this study was confirmed by induction of MIR399 2.1 | High Pi fertilization enhances susceptibility of expression and down-regulation of its target gene OsPHO2, a typical rice plants to the blast fungus M. oryzae response of plants to Pi starvation (Figure S3). To assess whether Pi supply has an effect on disease resistance in rice, we grew rice (Oryza sativa japonica ‘Tainung 67’) plants for 2.2 | MIR399 overexpression increases 15 days under different Pi regimes: high Pi, sufficient Pi, and low susceptibility to M. oryzae infection Pi (hereinafter high-Pi, sufficient-Pi, and low-Pi plants). Compared to plants that have been grown under sufficient Pi supply, high-Pi The rice miR399 family consists of 11 members (miR399a to and low-Pi plants accumulated higher and lower levels of free Pi in miR399k; miRBase v22.1). Of them, miR399f has identical mature leaves, respectively (Figure 1a). sequences in rice and Arabidopsis. Overexpression of miR399f Rice plants at the three- to four-leaf stage (high-, sufficient-, results in over-accumulation of Pi in leaves of Arabidopsis plants and low-Pi plants) were challenged with M. oryzae. At the time (Chiou et al., 2006). To further investigate the mechanisms by of inoculation, no major differences were observed in growth in which over-accumulation of Pi in rice plants enhances blast sus- plants grown under different Pi treatments (only some reduction ceptibility we generated transgenic rice (Tainung 67) lines over- in shoot fresh weight in both low- and high-Pi plants, compared expressing miR399f (hereinafter referred to as miR399 OE plants; to sufficient-Pi plants) (Figure S1a,b). On pathogen inoculation, Methods S1) which were then challenged with the rice blast high-Pi plants consistently displayed enhanced susceptibility to fungus. M. oryzae infection compared with sufficient- or low-Pi plants The accumulation of miR399 precursor transcripts, and concomi- (Figure 1b). The rice cultivars Maratelli and Saber were used as tant down-regulation of OsPHO2, was confirmed through successive reference cultivars in infection experiments (susceptible and generations of the miR399 OE lines supporting that the MIR399f resistant to M. oryzae, respectively). Scoring of symptom sever- transgene was functional and stably inherited (Figure S4a). At the ity was performed using the standard evaluation system (SES) adult stage, miR399 OE plants exhibited leaf tip necrosis in older ranging from 0 to 9 (0 = no symptom and 9 = highly suscep- leaves, a typical trait of plants accumulating a high Pi level (Figure S4b). tible). Whereas low- or sufficient-Pi plants had SES values of 1 Five independent miR399 OE lines harbouring a single copy of the and 2, respectively, the high-Pi plants showed SES values of 5 transgene inserted in their genome (Table S1) were selected for fur- (Figure 1b). Q uantification of diseased leaf area and fungal biomass ther studies. in the infected leaves further confirmed enhanced susceptibility to The transgenic miR399 OE lines accumulated mature miR399 M. oryzae in high-Pi plants (Figure 1c). These results demonstrate sequences at different levels, whereas OsPHO2 expression de- that, when growing Tainung 67 rice plants under high-Pi supply, creased in all the transgenic lines (Figure 3a, left and middle panels). these plants over-accumulate Pi in leaves and exhibit enhanced Compared to wild-type plants, miR399f OE plants accumulated a susceptibility to M. oryzae infection. higher level of Pi in their leaves (Figure 3a, right panel). Next, we investigated whether Pi treatment influences the de- Three-week-old miR399 OE plants at the three- to four-leaf velopment of blast disease not only in the rice cultivar Tainung 67, stage were grown under Pi-sufficient conditions and then chal- but also in other rice cultivars. To this end, rice cultivars with vary- lenged with M. oryzae. At 7 days post-inoculation (dpi), few blast ing degrees of blast susceptibility were grown under different symptoms were visible in leaves of wild-type plants, while leaves Pi regimes and then challenged with M. oryzae. The cultivars used of miR399 OE lines were severely affected (Figure 3b, left panel). were Nipponbare, Maratelli, Bomba, and Vialone Nano. For a com- Quantification of diseased leaf area confirmed susceptibility to parison, the resistant cultivar Saber, characterized for the presence M. oryzae infection in miR399 OE plants (Figure 3b, right panel). of the Pib resistance gene (Campos-Soriano et al., 2013), was in- These findings indicate that miR399 accumulation caused by cluded in these experiments. Under high-Pi conditions, all the sus- miR399 overexpression has a negative effect on blast resistance ceptible rice genotypes assayed in this study over-accumulated Pi in rice. in their leaves and showed increased susceptibility to blast infection The OsPR1a and OsPBZ1 genes are recognized as molecular (Figure 2). In contrast, high-Pi treatment had no effect on the disease marker genes for the induction of the rice defence response to phenotype of the resistant cultivar Saber (Pib gene), suggesting that M. oryzae infection (Midoh and Iwata, 1996; Agrawal et al., 2001). the Pib-mediated resistance mechanisms to blast are not broken by Accordingly, we examined the expression of these defence-related high-Pi treatment. Depending on the rice genotype, a small reduc- genes in leaves of miR399 OE lines and wild-type plants during tion in growth could be observed in both low-Pi and high-Pi plants M. oryzae infection. As expected, OsPR1a and OsPBZ1 expression compared with sufficient-Pi plants, as revealed by fresh weight and was induced in M. oryzae-inoculated wild-type plants compared to dry weight measurements (Figure S2). mock-inoculated plants (Figure 3c). Although OsPR1a and OsPBZ1 Under the experimental conditions assayed, low-Pi treatment expression was also activated by M. oryzae infection in miR399 OE had a minimal impact on blast disease in all the rice genotypes plants, they were induced to a lesser extent in transgenic plants than 558 | CAMPOS-SORIANO et al. F I G U R E 1 Susceptibility to infection by Magnaporthe oryzae in rice plants (Oryza sativa ‘Tainung 67’) grown under high-Pi supply. (a) Free Pi content in leaves of rice plants grown under low, sufficient, or high Pi for 15 days (three- to four-leaf stage). Bars represent mean ± SEM of four biological replicates with three plants per replicate. Comparisons have been made relative to the sufficient Pi condition (t test, *p < .05). (b) Disease symptoms at 7 days post-infection (dpi) of low-, sufficient-, or high-Pi treated plants. Representative results from one of three independent experiments that produced similar results are shown. Reference cultivars used were Saber (resistant) and Maratelli (susceptible). Standard evaluation system (SES) values are indicated on the right. (c) Percentage of diseased leaf area at 7 dpi (15 leaves per Pi treatment) (left panel). All the treatments were compared to the sufficient Pi supply (Wilcox test, **p ≤ .001). Fungal biomass was quantified at 7 dpi by quantitative PCR analysis using specific primers of M. oryzae Mo28S (right panel) and normalized to rice Ubiquitin 1 gene (eight biological replicates per Pi treatment). Comparisons have been made relative to the sufficient Pi supply (t test, **p ≤ .005, ***p ≤ .0001) wild-type plants during M. oryzae infection (this different induction grew plants (Tainung 67) for 15 days under low-, sufficient-, and was more evident for OsPR1a) (Figure 3c). Intriguingly, in the absence high-Pi conditions. The expression of OsPBZ1 and OsPR1a was de- of pathogen infection, the miR399 OE lines also exhibited a slight termined at different times after inoculation (24, 48, and 72 hours up-regulation of OsPBZ1 compared with wild-type plants (Figure 3c, post-inoculation [hpi]). OsPR1a and OsPBZ1 expression was in- right panel). A weaker induction of defence responses during infec- duced in M. oryzae-inoculated plants, but the level of induction of tion is consistent with the observed phenotype of disease suscepti- these genes differed among the Pi treatments (differences were bility in miR399 OE plants. clearly observed at 48 and 72 hpi; Figure 4). Thus, an increase in Knowing that miR399 OE plants over-accumulate Pi in their Pi supply is accompanied by a lower induction of OsPBZ1 during leaves, and that OsPR1a and OsPBZ1 expression showed a weaker infection of wild-type plants (Figure 4). Compared to sufficient-Pi induction in miR399 OE plants during M. oryzae infection, it plants, OsPR1a is also activated to a lower extent in high-Pi plants was of interest to assess whether Pi treatment has an effect on (at 48 hpi, although similar levels of OsPR1a expression occur in OsPR1a and OsPBZ1 expression in wild-type plants. For this, we high- and sufficient-Pi plants at 72 hpi). A weaker induction of CAMPOS-SORIANO et al. | 559 F I G U R E 2 Susceptibility to infection by Magnaporthe oryzae in different rice genotypes grown under high-Pi supply. Free Pi content in leaves of rice plants grown under low, sufficient, or high Pi for 15 days (three- to four-leaf stage, upper panel) of the indicated rice genotypes. Bars represent mean ± SEM of four biological replicates with three plants per replicate. Comparisons have been made relative to the sufficient Pi condition (t test, *p < .05, **p < .005, ***p < .0001) Representative images of disease symptoms at 7 days post-inoculation with M. oryzae spores (lower panel) defence gene expression in high-Pi wild-type rice plants during agriG​O; Du et al., 2010) and visualized by REVIGO (Supek et al., M. oryzae infection is in agreement with previous observations 2011). This analysis revealed statistically enriched terms in biolog- on susceptibility to M. oryzae infection in these plants (see ical processes, cellular components, and molecular functions that Figure 1b; Tainung 67). However, the higher induction of OsPBZ1 were likely to be regulated by miR399 in rice. In the set of up- and OsPR1a that occurs in low-Pi plants relative to sufficient-Pi regulated genes, the most abundant terms in the biological func- plants (Figure 4) led to only a slight increase in disease resistance tion category were “metabolism” (e.g., “phosphorus metabolism”), (see Figure 1a). “response to stimulus” (e.g., “response to biotic stimulus”), and Collectively, the results obtained on blast disease assays in “post-translational protein modification” (Figures 5a and S5). The wild-type plants grown under high Pi supply, together with those GO terms over-represented in down-regulated genes clustered obtained in miR399 OE plants, support the fact that Pi accumula- in the categories of “metabolism” (primary metabolism, phos- tion negatively affects defence gene expression, hence blast re- phorus metabolism, protein metabolism), “response to stimulus”, sistance, in rice. “phosphorylation”, and “post-translational protein modification” (Figures 5b and S6). The “cellular component” category from up- regulated genes contained the terms “integral to membrane” and 2.3 | Transcript profiling of miR399 OE rice plants “nucleus”, whereas down-regulated genes were enriched with the GO term “nucleus” (Figure S7). To better understand the molecular mechanisms underlying blast AgriGO analysis also revealed that the most abundant sub- susceptibility in miR399 OE rice plants, we performed transcriptome categories in the molecular function category included “protein analysis of miR399 OE lines and wild-type plants. Differentially ex- kinase activity” and “ATP binding”, in both up-regulated and down- pressed genes (DEGs) were identified based on significance level regulated genes (Figures S8 and S9), whereas genes with “tran- (p ≤ .05) and fold change (log2 fold change ≥0.5 or ≤0.5 as the thresh- scription factor activity” were highly represented among the old for up-regulated and down-regulated genes, respectively). Using up-regulated genes in miR399 OE plants (Figure S8). The “zinc ion these criteria, a total of 965 genes were found to be differentially binding” and “calcium ion binding” subcategories were specifically expressed in miR399 OE plants relative to wild-type plants, of which enriched in up-regulated genes (Figure S8), while “iron binding” 690 genes were up-regulated and 275 genes were down-regulated genes were enriched in down-regulated genes (Figure S9). (Table S2). A heat map analysis of genes specifically annotated in the subcat- Gene ontology (GO) enrichment analysis of DEGs in miR399 egories “protein phosphorylation and ATP binding” and “regulation of OE plants was carried out by AgriGO (http://bioin​fo.cau.edu.cn/ transcription” is presented in Figure S10 (left and middle panel; genes 560 | CAMPOS-SORIANO et al. F I G U R E 3 Susceptibility to infection by the rice blast fungus Magnaporthe oryzae in miR399 overexpressing (OE) rice plants. (a) Accumulation of mature miR399 sequences (left panel) and OsPHO2 transcripts (middle panel) in leaves of wild-type (Tainung 67; WT) and five independent miR399 OE lines (independent homozygous lines, L2, L4, L20, L31, and L32) determined by small RNA northern blot and quantitative reverse transcription PCR analysis, respectively. Plants were grown under sufficient-Pi conditions. Free Pi accumulation in miR399 OE lines and WT plants (right panel). Bars represent mean ± SEM of four biological replicates with three plants per replicate (t test, *p ≤ .05, **p < .005). (b) Susceptibility of miR399 OE plants to M. oryzae infection. Five independent homozygous lines (six plants each line) were assayed in each of four independent experiments with similar results (results for three miR399 OE lines are shown, L2, L4, and L20, similar results were obtained for the other lines). Reference cultivars: Saber (resistant) and Maratelli (susceptible). Right panel, diseased leaf area in infected leaves at 7 days post-inoculation with M. oryzae spores (six leaves per line, t test, *p ≤ .05, **p ≤ .005; miR399 OE lines versus WT). (c) Accumulation of OsPR1a and OsPBZ1 transcripts in leaves of mock-inoculated and M. oryzae-inoculated wild-type and miR399 OE plants (L2, L4, L20) at 48 hours post-inoculation. Bars represent mean of three biological replicates (three pooled plants in each replicate) ± SEM (t test, *p ≤ .05, **p < .005, infected miR399 OE lines versus infected WT) CAMPOS-SORIANO et al. | 561 obtained by RNA-Seq analysis, the expression of these genes was up-regulated in miR399 OE plants compared to wild-type plants. 2.4 | miR399 overexpression influences the expression of genes involved in protein phosphorylation and transcriptional regulation The expression of a vast array of genes involved in protein phosphoryla- tion was misregulated in miR399 OE plants (up-egulation and down- regulation) (Figure S10 and Table S3). Among the up-regulated genes, we identified genes representing different families of receptor protein kinases and receptor-like protein kinases, including wall-associated kinases (WAKs; OsWAK36, OsWAK87, OsWAK41, OsWAK98), Ser/ Thr receptor protein kinases (e.g., the PR5K receptor kinase) lectin-like receptor protein kinases, and leucine-rich repeat receptor protein kinases (Table S3). WAKs are known to play a key role in basal defence against the rice blast fungus (Delteil et al., 2016). Regarding the Ser/Thr protein kinase receptor PR5K, it contains an extracellular domain struc- turally related to pathogenesis-related PR5 proteins involved in disease resistance (Wang et al., 1996). The receptor kinase LecRK-I.9 has been reported to regulate jasmonic acid (JA) signalling and disease resist- ance in Arabidopsis (Balagué et al., 2017). The observed up-regulation of receptor protein kinases for which a positive role in defence responses has been described is in apparent contradiction to the phenotype of blast susceptibility observed in miR399 OE plants. In the set of down-regulated genes in miR399 OE plants, there were three phytosulfokine receptor genes (members of the leucine-rich repeat family of receptor kinases) (Figure S10, code PSKR-1, PSKR-2, PSKR-3; Table S3). Binding of phytosulfokine pep- tides to phytosulfokine receptors differentially modulates Arabidopsis immune responses (Sauter, 2015). The expression of genes belong- ing to different families of protein kinases was also misregulated in miR399 OE plants (Figure S10 and Table S3). They included Ser/Thr protein kinases (code KIN), tyrosine protein kinases (code, PKDCC), F I G U R E 4 Expression of the defence marker genes OsPBZ1 and and calcium-dependent protein kinase (code CDPK) genes. Our tran- OsPR1a in mock- or Magnaporthe oryzae-inoculated leaves of low-, sufficient-, or high-Pi wild-type (Tainung 67; WT) plants. Quantitative scriptome analysis also revealed that up to seven adenosine triphos- reverse transcription PCR was carried out at the indicated times after phatase genes were up-regulated in miR399 OE plants. Knowing that inoculation using the rice Ubiquitin 5 gene as the internal control. Bars miR399 overexpression was associated with an increase in Pi content, represent means of three biological replicates, each one from a pool of the observed alterations in the expression of genes involved in protein three different plants ± SEM (t test, *p ≤ .05, **p < .005, ***p < .0001) phosphorylation and ATP binding might well be the consequence of Pi accumulation in leaves of miR399 OE plants. shown in the heat map are listed in Tables S3 and S4). Intriguingly, Moreover, miR399 overexpression regulates the expression of a defence-related genes were found among the set of both up-regulated considerable number of TFs belonging to several TF families, most and down-regulated genes in miR399 OE plants (Figure S10, right of them being up-regulated in miR399 OE plants (Figure S10 and panel, and Table S5). These findings are further discussed below. Table S4). They included WRKY, AP2 (APETALA2)-related (e.g., ethylene- The expression of genes identified by RNA-Seq analysis of responsive TFs), NAC (NAM, no apical meristem; ATAF1; and CUC2, miR399 OE plants was further investigated by quantitative reverse cup-shaped cotyledon), Myb (myeloblastosis), bZIP (basic leucine transcription PCR (RT-qPCR). Genes that were classified into dif- zipper), C2H2 (C2H2 zinc finger), and ZF1 (zinc finger) TF genes. It is ferent functional categories were selected for this study, including generally assumed that WRKY TFs are important regulators of stress genes involved in defence responses, diterpenoid phytoalexin bio- responses in plants, including rice blast resistance (Wei et al., 2013; synthesis, receptor protein kinase genes, protein kinase genes, and Phukan et al., 2016). It should be noted that up to 22 WRKY TFs are transcription factor (TF) genes (Figure 6). In agreement with results up-regulated in miR399 OE plants, including OsWRKY45 (Figure 6 and 562 | CAMPOS-SORIANO et al. F I G U R E 5 Gene ontology (GO) enrichment analysis of differentially expressed genes in miR399 overexpressing (OE) plants relative to wild- type plants. The top GO terms enriched in miR399 OE plants were represented by using REVIGO after reducing redundancy (http://revigo.irb.hr/). (a) and (b) represent up-regulated and down-regulated genes, respectively. Circles represent GO terms and those clustered closer to each other represented similar GO terms. Circle colours (blue to yellow) represent the degree of GO enrichment (p value) and size is proportional to the frequency of the GO term in the GO database (larger and smaller discs represent more general and more specific terms, respectively) Table S4). OsWRKY45 has been reported to play a crucial role in resis- Because the miR399 OE rice plants exhibited enhanced suscep- tance to blast infection, and its overexpression enhances resistance tibility to M. oryzae infection, the observed expression of defence- to infection (Shimono et al., 2007). As previously observed for recep- related genes (e.g., PR, phytoalexin biosynthesis genes) in these plants tor protein kinases involved in defence responses, an up-regulation of appears not to be effective enough to arrest pathogen infection. This OsWRKY45 in miR399 OE plants is in apparent contradiction to the observation posed intriguing questions related to regulation of de- phenotype of disease susceptibility in these plants. WRKY TFs have fence gene expression in miR399 OE plants. We reasoned that miR399 been also shown to regulate tolerance to Pi starvation in Arabidopsis overexpression, while causing a basal expression of some defence- and rice (Wang et al., 2014; Dai et al., 2016). related genes under noninfection conditions, might interfere with the activation of defence responses during pathogen infection. To test this possibility, we monitored defence gene expression in miR399 OE 2.5 | An increase in Pi content compromises the and wild-type plants that had been inoculated with M. oryzae or mock- basal defence of rice plants during pathogen infection inoculated (48 hpi). Compared to wild-type plants, a weaker induction of defence gene expression occurred in miR399 OE plants in response to As previously mentioned, RNA-Seq analysis revealed misregulation of M. oryzae infection, including OsPR10b, OsCHIT1, OsLAC19, OsWAK46, certain defence-related genes in miR399 OE plants in the absence of OsMAPK5, and OsWRKY45 (Figure 7). Similarly, a weaker response was pathogen infection, some of them being up-regulated in these plants observed in genes involved in diterpenoid phytoalexin biosynthesis (e.g., members of the PR1, PR5, PR8, and PR10 families of PR genes) (OsCPS2, OsKSL4, OsKSL7, Oryzalexin D synthase, Oryzalexin E synthase, (Figures 6 and S10; Table S5). Genes involved in protection against OsMAS1). Whether miR399 OE plants fail to accumulate diterpenoid oxidative stress and/or cell wall lignification were also misregulated in phytoalexins during M. oryzae infection remains to be investigated. miR399 OE plants (e.g., peroxidase and laccase genes) (Figures 6 and S10; The observation that miR399 overexpression leads to an in- Table S5). Peroxidases and laccases contribute to plant cell-wall lig- crease in Pi content and repression of the pathogen-inducible host nification by catalysing the polymerization of monolignols (Miedes defence response prompted us to investigate whether genes for et al., 2014). Peroxidases also participate in maintenance of the cellular which a weaker induction occurred during infection in miR399 OE redox balance to protect against oxidative stress (Almagro et al., 2009). plants were also induced at a lower extent in rice plants that have Unexpectedly, RNA-Seq analysis of miR399 OE plants showed a basal been grown under high-Pi conditions. In response to M. oryzae in- expression level of several genes involved in diterpenoid phytoalexin fection, all the defence-related genes here examined (OsPR10b, biosynthesis, these genes being responsible for the production of OsCHIT1, OsLAC19, OsMAPK5, OsWRKY45, and OsWAK36) had a major rice phytoalexins (momilactones, oryzalexin A, B, C, and D, lower induction in high-Pi plants compared to sufficient-Pi wild-type oryzalexin E, and phytocassenes) (Yamane, 2013). plants (Figure 7; fold-induction is presented). CAMPOS-SORIANO et al. | 563 F I G U R E 6 Expression of differentially expressed genes identified by RNA-Seq analysis. Transcript levels were determined by quantitative reverse transcription PCR in leaves of wild-type (WT) and miR399 overexpressing lines (lines L4 and L20, similar results were obtained for L2). Plants were grown under sufficient-Pi conditions. The expression values were normalized to the rice Ubiquitin 1 gene. Gene-specific primers sequences with its gene locus ID and heat maps code (from Figure S10) are presented in Table S6. Bars represents means of three biological replicates, each one from a pool of three different plants, ±SEM. Comparisons have been made relative to the WT (t test, *p ≤ .05, **p ≤ .005) Collectively, the results presented in this study demonstrate that infection. These findings, together with the observation that defence an increase in Pi level caused either by miR399 overexpression or gene expression decreases when increasing Pi supply, support the fact treatment with high-Pi represses the pathogen-inducible host defence that Pi over-accumulation (high-Pi, miR399 OE plants) compromises response. This, in turn, would negatively affect resistance to M. oryzae the immune system in rice, hence fostering disease susceptibility. 564 | CAMPOS-SORIANO et al. F I G U R E 7 Induction of defence-related genes in rice plants accumulating Pi in leaves on inoculation with Magnaporthe oryzae spores. (a) miR399 overexpressing (OE) plants grown under sufficient-Pi conditions. Results obtained on lines L4 and L20 are shown (similar results were obtained for line L2). (b) Wild-type (WT) plants that have been grown under high Pi supply. The expression values were normalized to the rice Ubiquitin 5 gene. Bars represent means of three biological replicates, each one from a pool of three different plants, ±SEM. In all the cases, the expression level in mock-inoculated plants was set to 1.0 3 | D I S CU S S I O N by high Pi fertilization enhances blast susceptibility in different rice cultivars, (e.g., Tainung 67, Nipponbare, Maratelli, Bomba, and Vialone Here we provide evidence that Pi, when in excess, is an important Nano). Equally, miR399f overexpression causes Pi accumulation and factor in determining susceptibility to infection by the foliar patho- enhances blast susceptibility. Disease susceptibility in rice plants ac- gen M. oryzae in rice. We show that an increase in Pi content caused cumulating Pi in leaves was associated with a weaker induction of CAMPOS-SORIANO et al. | 565 defence gene expression during pathogen infection. This finding in- and responds to Pi excess by activating genes typically associated dicates that these plants are not able to mount an effective defence with the plant response to pathogen infection. A tempting hypothe- response, which is in agreement with their phenotype of blast sus- sis is that high Pi is perceived by the plant as a stressful situation and ceptibility. Contrary to what is observed in susceptible rice cultivars, that this sensing triggers a basal expression of defence-related genes Saber plants did not develop disease symptoms regardless of Pi supply, by yet unknown signalling mechanisms in the absence of pathogen suggesting that resistance triggered by the Pib resistance gene is not infection. Clearly, the observed weaker induction of M. oryzae- affected by high-Pi supply. inducible defence gene expression in Pi-accumulating rice plants, Pi excess might reduce the plant's ability to overcome pathogen and concomitant blast susceptibility, indicates that these plants infection in different ways. On the one hand, Pi excess may be re- respond in a less effective manner to pathogen infection, while sup- sponsible for metabolic changes in the host plant that jeopardize the porting that there is an interplay between Pi- and pathogen-induced plant defence response. Along with this, our transcriptome analysis signal transduction pathways in rice. Further investigation is needed showed that miR399 overexpression causes important perturbations to understand how signals coming from the two types of stresses, in genes involved in metabolic processes, in particular lipid and carbo- Pi excess and pathogen infection, are integrated during pathogen hydrate metabolism. Another possibility, not excluding the previous infection. Here, it is worth mentioning that whereas transcriptional one, is that fungal pathogenicity itself is affected by Pi availability reprogramming of gene expression in response to Pi starvation is and that the fungus modifies its pathogenicity programme in order widely described (Misson et al., 2005; Wasaki et al., 2006; Oono to adapt to a situation of high Pi in the host tissue. An increase in et al., 2013; Secco et al., 2013; Gho et al., 2018), the transcriptional Pi level might create a more favourable environment for pathogen response to Pi excess remains largely unknown. Very recently, a growth that enables the pathogen to improve nutrient acquisition phosphate transporter from rice (OsPT8) has been reported to nega- from the host plant, thereby promoting pathogenicity. Alternatively, tively regulate blast resistance, thus reinforcing the notion of cross- high Pi content might also stimulate the production of pathogen vir- talk between Pi homeostasis and immune signalling pathways in rice ulence factors that suppress host defence mechanisms. In the case (Dong et al., 2019). of nitrogen-induced susceptibility to rice blast, it was reported that In addition, transgenic expression of a phytoplasma effector alterations in some metabolites (e.g., glutamine) are responsible for (SAP11AYWB) in Arabidopsis was found to increase miR399 accumu- increased fungal pathogenicity (Huang et al., 2017). At present, how- lation, thereby increasing Pi level (Lu et al., 2014). During infection of ever, it is not known whether an increase in Pi content in rice leaves SAP11AYWB Arabidopsis plants with the bacterial pathogen Pseudomonas has an effect on M. oryzae virulence. On the other hand, studies in syringae, the induction of defence responses was suppressed and Arabidopsis have shown that Pi content influences the structure of the SAP11AYWB plants exhibited susceptibility to bacterial infection the root-associated bacterial (Castrillo et al., 2017; Finkel et al., 2019) (Lu et al., 2014). In other studies, cross-talk between Pi starvation and fungal communities (Fabianska et al., 2019). It was proposed that and salt and drought stress signalling pathways, or hormone-signal- changes in root fungal communities of Arabidopsis plants correlate ling pathways, has been described (Song and Liu, 2015; Abbas Khan with concurrent changes in the plant phosphate starvation response, et al., 2016; Baek et al., 2017; Castrillo et al., 2017). Knowing that the suggesting that P-deprived and P-replete plants select different fun- defence-related hormones JA and ethylene (ET) function as signalling gal taxa in their roots (Fabianska et al., 2019). Whether Pi content hormones in the rice defence against M. oryzae (Nasir et al., 2018), a has an effect on potential rice pathogens needs to be further inves- major future challenge is to determine whether Pi content affects de- tigated. We also show that, even though the low-Pi plants have acti- fence hormone signalling in rice for the control of immune responses. vated the phosphate starvation response, low-Pi treatment does not Collectively, these observations suggest that, although a function for appear to affect blast severity in a substantial manner, at least under miR399 in the two stress signalling systems, Pi signalling and immu- the experimental conditions here assayed. There is the possibility that nity, can be postulated, we have yet to decipher the molecular mech- activation of the phosphate starvation response allows the plant to anisms of this cross-talk. cope with the low-Pi stress condition imposed in this study, and more Phosphorus is a key component of many important biological stressful conditions (e.g., lower Pi supply, longer periods of low Pi molecules, including ATP, which exists not only in the cytoplasm, treatment) might be needed to observe a differential blast phenotype but also in the extracellular matrix. Of interest, plants are capable in low-Pi rice plants. of sensing and responding to extracellular ATP during pathogen Another finding of this study is that, under normal growth con- infection, a situation in which ATP is perceived as a damage-associated ditions, the miR399 OE plants show a basal expression of some molecular pattern (DAMP) for cellular damage caused during defence-related genes (e.g., OsWRKY45, diterpenoid phytoalexin pathogen colonization (Tanaka et al., 2014). In this manner, extra- biosynthesis genes) in the absence of pathogen infection. On patho- cellular ATP can be considered as a signalling molecule for the ac- gen challenge, however, the basal level of defence gene expression tivation of Arabidopsis defence responses (Cao et al., 2014). More that is observed in Pi-accumulating plants (both miR399 OE and recently, extracellular ATP has been shown to play a role in the wild-type plants grown under high-Pi supply) does not seem to be JA-induced defence response of Arabidopsis through the direct ac- effective enough to arrest pathogen infection. These observations tivation of JA signalling (but not via JA biosynthesis) (Tripathi et al., raise a number of interesting questions on how the plant perceives 2018; Jewell et al., 2019). Evidence for a role of extracellular ATP 566 | CAMPOS-SORIANO et al. F I G U R E 8 Proposed model for the role of miR399 and Pi homeostasis in the defence response to Magnaporthe oryzae. Under no infection conditions, high-Pi supply in wild-type plants and MIR399 overexpression (OE) entails Pi overaccumulation in rice leaves. This would trigger a state of nutritional stress with metabolic alterations and changes in ATP signalling, which ultimately would activate the expression of stress- related genes, such as those involved in protein phosphorylation, transcription factors, peroxidases and laccases, and phytoalexin biosynthesis, as well as PR genes. On pathogen challenge, a weaker induction of these stress-related genes would occur in high Pi-treated plants and miR399 OE plants compared to wild-type plants with a sufficient Pi supply. This, in turn, would lead to susceptibility to M. oryzae infection. Arrows and blunt ends, positive and negative regulation, respectively. Arrows with broken lines, unknown interlocked regulatory mechanisms as signalling molecule for the expression of defence responses in On the contrary, cross-talk between Pi and other nutrients is well rice is, however, lacking. documented in plants and miRNAs have been shown to be involved Based on the results obtained in our transcriptome analy- in cross-talk between nutrient deficiencies in Arabidopsis (Liang et al., sis of miR399 OE plants, a working model summarizing the im- 2015). In particular, miR399 has been proposed to participate in the pact of miR399 overexpression (and Pi accumulation) in regulating regulation of multiple nutrient starvation responses in rice (Hu et al., defence responses to pathogen infection in rice is presented in 2015). Under this scenario, stress imposed by high Pi might also ham- Figure 8, which includes processes related to pathogen recognition, per the coordination of homeostatic pathways of Pi and other nutri- signalling processes, and defence reactions. According to this model, ents, and Pi excess caused by miR399 overexpression or high Pi supply miR399-mediated perturbations in host gene expression would might alter other nutrients’ accumulation. This nutritional imbalance affect diverse physiological processes in rice plants under nonin- would trigger an additional stress to the host plant that would impair fection and infection conditions. Thus, miR399 overexpression (as the optimum defence response to pathogen infection (Figure 8). This well as growing rice plants under high Pi supply) would provoke a piece of information will be useful in future studies to understand how stressful situation in the plant that would have pleiotropic effects plants integrate Pi signalling with immune responses. on metabolic processes and defence responses, and possibly also The Arabidopsis miR399f has been reported to function as a pos- ATP signalling. The observation that miR399 overexpression alters itive regulator in tolerance to salt stress, while negatively regulat- the expression of diverse protein kinases and transcription factors ing drought stress, through the control of two novel target genes potentially involved in pathogen recognition and/or defence signal in Arabidopsis, the ABF3 transcription factor and CSP41b (unknown transduction (e.g., receptor protein kinases, Ser/Thr kinases, among function) genes (Baek et al., 2016). Although the results presented others) favours this possibility. These alterations would decrease the here indicate that miR399 overexpression is accompanied by ability of the host plant to detect the invading pathogen and respond down-regulation of OsPHO2 in miR399 OE plants, the possibility in a timely and appropriate manner, leading to enhanced susceptibil- that miR399f directs cleavage of still unknown target genes in rice ity to M. oryzae infection. should not be discarded. CAMPOS-SORIANO et al. | 567 Collectively, the results presented here support the finding that wild-type and miR399 OE plants. Each biological replicate consisted Pi accumulation in rice leaves negatively affects basal resistance to of leaves from 15 plants. Two independent miR399 OE lines and M. oryzae in rice by reducing the plant’s ability to cope with pathogen three biological replicates for each genotype were examined (see infection. Here, it is worth emphasizing that fungicides are widely Methods S1 for details on RNA-Seq analysis). The RNA sequence used to protect rice plants against blast disease, which causes se- data were deposited at the National Center for Biotechnology rious environmental problems. Indeed, rice blast is considered the Information (NCBI) Gene Expression Omnibus (GEO repository (ac- most important fungus-caused disease in plants in terms of scientific cession number GSE13​7735). and economic relevance (Dean et al., 2012). Pi fertilization should then be considered on a cost–benefit basis in rice farming. The re- sults presented here might lay a foundation for rationally optimizing 4.5 | Gene expression analyses fertilizer and pesticide use in rice production. Total RNA was extracted from rice leaves using the TRIzol reagent (Invitrogen). For northern blot analysis, total RNAs were fractionated 4 | E X PE R I M E NTA L PRO C E D U R E S in a 17.5% denaturing polyacrylamide gel containing 8 M urea, trans- ferred to nylon membranes, and hybridized with [γ32P]ATP-labelled 4.1 | Plant material oligonucleotides (Table S6). Hybridization signals were detected using a phosphorimager (Bio-Rad). Synthetic RNA oligonucleotides Rice plants were grown at 28 ºC with a 14 hr:10 hr light/dark cycle. were used as size markers. Transgenic rice (cultivar Tainung 67) plants were produced by RT-qPCR was performed in optical 96-well plants using SYBR Agrobacterium-mediated transformation of embryogenic calli derived Green. Primers were designed using Primer-BLAST (https​://www.ncbi. from mature embryos (Sallaud et al., 2003). Transgene copy number was nlm.nih.gov/tools/​primer-blast/​). Accumulation of mature miR399 assessed by PCR on genomic DNA as previously described (Yang et al., sequences was determined by stem-loop RT-qPCR. Primers used for 2005). Primers are shown in Table S6. The susceptible rice cultivars RT-qPCR and stem-loop RT-qPCR are listed in Table S6. Further details Nipponbare, Maratelli, Vialone Nano, and Bomba and the resistant cul- on RT-qPCR and stem-loop RT-qPCR can be found in Methods S1. tivar Saber harbouring the Pib resistance gene were used in this work. AC K N OW L E D G E M E N T S M.B.P. was funded by a “la Caixa” scholarship for PhD studies in 4.2 | Infection assays Spanish universities. We thank J. Freixanet for assistance in parts of this work. This research was supported by the Ministerio de Ciencia, Infection assays with the rice blast fungus M. oryzae (strain Innovación y Universidades (MCIU)-Agencia Estatal de Investigación Guy 11) were carried out on soil-grown rice plants at the three- (AEI) y el Fondo Europeo de Desarrollo Regional (FEDER) (BIO2015- to four-leaf stage. Further experimental details can be found in 67212-R and RTI2018-101275-B-100) and the Academia Sinica, Methods S1. The percentage of diseased leaf area was determined Taiwan (AS-SS-106-03-3). We also acknowledge support from the at 7 dpi with M. oryzae spores by using the APS Assess 2.0 program CERCA Programme (“Generalitat de Catalunya”) and the “Severo (Lamari, 2008). Fungal biomass on infected leaves was quantified by Ochoa Programme for Centres of Excellence in R&D” 2016-2019 quantitative PCR (qPCR) using specific primers for the M. oryzae 28S (SEV-2015-0533). The authors declare that there is no conflict of DNA gene (Qi and Yang, 2002). interest regarding the publication of this paper. DATA AVA I L A B I L I T Y S TAT E M E N T 4.3 | Pi treatment and measurement of Pi content The data that support the findings of this study are openly available in the NCBI-GEO repository at https​://www.ncbi.nlm.nih.gov/geo/, To assess the effect of Pi supply, rice plants were grown for 21 days reference number GSE13​7735. in a mixture of 50% peat and vermiculite (>50 mg/L phosphorus) and 50% inorganic quartz sand substrate. During the last 15 days, ORCID plants were fertilized with modified Hoagland half-strength solution Blanca San Segundo https://orcid.org/0000-0001-7409-3172 (2.5 mM Pi for high-Pi, 0.25 mM Pi for sufficient-Pi, and no Pi for low-Pi supplies). The Pi content of rice leaves was determined as pre- REFERENCES viously described (Ames, 1966; Versaw and Harrison, 2002). Abbas, K.G., Vogiatzaki, E., Glauser, G. and Poirier, Y. (2016) Phosphate deficiency induces the jasmonate pathway and enhances resistance to insect herbivory. Plant Physiology, 171, 632–644. Agrawal, G.K., Rakwal, R., Jwa, N.S. and Agrawal, V.P. (2001) Signalling 4.4 | Library preparation and RNA-Seq analysis molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: a model illustrating components participating during Total RNA was extracted using the Maxwell 16 LEV Plant RNA defence/stress response. Plant Physiology and Biochemistry, 39, 1095–1103. Kit (Promega). Libraries were prepared from leaves of 3-week-old 568 | CAMPOS-SORIANO et al. Almagro, L., Gómez Ros, L.V., Belchi-Navarro, S., Bru, R., Ros Barceló, A. Dai, X., Wang, Y. and Zhang, W.H. (2016) OsWRKY74, a WRKY tran- and Pedreño, M.A. (2009) Class III peroxidases in plant defence reac- scription factor, modulates tolerance to phosphate starvation in rice. tions. Journal of Experimental Botany, 60, 377–390. Journal of Experimental Botany, 67, 947–960. Ames, B.N. (1966) Assay of inorganic phosphate, total phosphate and Dean, R., Van Kan, J.A.L., Pretorius, Z.A., Hammond-Kosack, K.E., phosphatases. Methods in Enzymology, 8, 115–118. Di Pietro, A., Spanu, P.D. et al. (2012) The top 10 fungal pathogens in Aung, K., Lin, S.I., Wu, C.C., Huang, Y.T., Su, C.L. and Chiou, T.J. (2006) molecular plant pathology. Molecular Plant Pathology, 13, 414–430. pho2, a phosphate overaccumulator, is caused by a nonsense Delteil, A., Gobbato, E., Cayrol, B., Estevan, J., Michel-Romiti, C., Dievart, mutation in a microRNA399 target gene. Plant Physiology, 141, A. et al. (2016) Several wall-associated kinases participate positively 1000–1011. and negatively in basal defence against rice blast fungus. BMC Plant Baek, D., Chun, H.J., Kang, S., Shin, G., Park, S.J., Hong, H. et al. (2016) Biology, 16, 17. A role for Arabidopsis miR399f in salt, drought, and ABA signaling. Ding, J., Jia, J., Yang, L., Wen, H., Zhang, C., Liu, W., et al. (2004) Validation of a Molecules and Cells, 39, 111–118. rice specific gene, Sucrose Phosphate Synthase, used as the endogenous Baek, D., Chun, H.J., Yun, D. and Kim, M.C. (2017) Cross-talk between reference gene for qualitative and real-time quantitative PCR detection phosphate starvation and other environmental stress signaling path- of transgenes. Journal of Agricultural and Food Chemistry, 52, 3372–3377. ways in plants. Molecules and Cells, 40, 697–705. Dong, Z., Li, W., Liu, J., Li, L., Pan, S., Liu, S. et al. (2019) The rice phos- Balagué, C., Gouget, A., Bouchez, O., Souriac, C., Haget, N., phate transporter protein OsPT8 regulates disease resistance and Boutet-Mercey, S. et al. (2017) The Arabidopsis thaliana lectin recep- plant growth. Scientific Reports, 9, 5408. tor kinase LecRK-I.9 is required for full resistance to Pseudomonas Du, Z., Zhou, X., Ling, Y., Zhang, Z. and Su, Z. (2010) agriGO: a GO anal- syringae and affects jasmonate signaling. Molecular Plant Pathology, ysis toolkit for the agricultural community. Nucleic acids research, 8, 937–948. 38(suppl_2), W64–W70. Baldrich, P. and San Segundo B. (2016) MicroRNAs in rice innate immu- Fabianska, I., Gerlach, N., Almario, J. and Bucher, M. (2019) Plant- nity. Rice, 9, 1–9. mediated effects of soil phosphorus on the fungal microbiota in Baldrich, P., Campo, S., Wu, M.T., Liu, T.Z., Hsing, Y.I.C. and San, S.B. Arabidopsis thaliana. New Phytologist, 221, 2123–2137. (2015) MicroRNA-mediated regulation of gene expression in the re- Finkel, O.M., Salas-González, I., Castrillo, G., Spaepen, S., Law, T.F., sponse of rice plants to fungal elicitors. RNA Biology, 12, 847–863. Pereira LimaTeixeira, P.J. et al. (2019) The effects of soil phosphorus Ballini, E., Nguyen, T.T.T. and Morel, J.B. (2013) Diversity and genetics of content on plant microbiota are driven by the plant phosphate star- nitrogen-induced susceptibility to the blast fungus in rice and wheat. vation response. PLoS Biology, 17, e3000534. Rice, 6, 1–13. Gho, Y.S., An, G., Park, H.M. and Jung, K.H. (2018) A systemic view of phos- Bigeard, J., Colcombet, J. and Hirt, H. (2015) Signaling mechanisms in phate starvation-responsive genes in rice roots to enhance phosphate pattern-triggered immunity (PTI). Molecular Plant, 8, 521–539. use efficiency in rice. Plant Biotechnology Reports, 12, 249–264. Boller, T. and He, S.Y. (2009) Innate immunity in plants: An arms race Ham, B., Chen, J., Yan, Y. and Lucas, W.J. (2018) Insights into plant phos- between pattern recognition receptors in plants and effectors in mi- phate sensing and signalling. Current Opinion in Biotechnology, 49, 1–9. crobial pathogens. Science, 324, 742–744. Hinsinger, P., Betencourt, E., Bernard, L., Brauman, A., Plassard, C., Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M., Dunoyer, Shen, J. et al. (2011) P for two, sharing a scarce resource: Soil phos- P., Yamamoto, Y.Y., Sieburth, L. et al. (2008) Widespread trans- phorus acquisition in the rhizosphere of intercropped species. Plant lational inhibition by plant miRNAs and siRNAs. Science, 320, Physiology, 156, 1078–1086. 1185–1190. Hsieh, L.C., Lin, S.I., Shih, A.C.C., Chen, J.W., Lin, W.Y., Tseng, C.Y. et al. Campo, S., Peris-Peris, C., Siré, C., Moreno, A.B., Donaire, L., Zytnicki, (2009) Uncovering small RNA-mediated responses to phosphate de- M. et al. (2013) Identification of a novel microRNA (miRNA) from rice ficiency in Arabidopsis by deep sequencing. Plant Physiology, 151, that targets an alternatively spliced transcript of the Nramp6 (Natural 2120–2132. resistance-associated macrophage protein 6) gene involved in patho- Hu, B., Zhu, C., Li, F., Tang, J., Wang, Y., Lin, A. et al. (2011) LEAF gen resistance. New Phytologist, 199, 212–227. TIP NECROSIS1 plays a pivotal role in the regulation of multiple Campos-Soriano, L., Valé, G., Luppotto, E. and San Segundo, B. (2013) phosphate starvation responses in rice. Plant Physiology, 156, Investigation of rice blast development in susceptible and resistant 1101–1115. rice cultivars using a gfp-expressing Magnaporthe oryzae isolate. Hu, B., Wang, W., Deng, K., Li, H., Zhang, Z., Zhang, L. et al. (2015) Plant Pathology, 62, 1030–1037. MicroRNA399 is involved in multiple nutrient starvation responses Cao, Y., Tanaka, K., Nguyen, C.T. and Stacey, G. (2014) Extracellular ATP in rice. Frontiers in Plant Science, 6, 188. is a central signaling molecule in plant stress responses. Current Huang, H., Nguyen Thi Thu, T., He, X., Gravot, A., Bernillon, S., Ballini, E. Opinion in Plant Biology, 20, 82–87. et al. (2017) Increase of fungal pathogenicity and role of plant gluta- Castrillo, G., Teixeira, P.J.P.L., Paredes, S.H., Law, T.F., De Lorenzo, L., mine in nitrogen-induced susceptibility (NIS) to rice blast. Frontiers in Feltcher, M.E. et al. (2017) Root microbiota drive direct integration of Plant Science, 8, 265. https​://doi.org/10.3389/fpls.2017.00265​. phosphate stress and immunity. Nature, 543, 513–518. Jewell, J.B., Sowders, J.M., He, R., Willis, M.A., Gang, D.R. and Tanaka, Cook, D.E., Mesarich, C.H. and Thomma, B.P. (2015) Understanding plant K. (2019) Extracellular ATP shapes a defence-related transcriptome immunity as a surveillance system to detect invasion. Annual Review both independently and along with other defence signaling path- of Phytopathology, 53, 541–563. ways. Plant Physiology, 179, 1144–1158. Couto, D. and Zipfel, C. (2016) Regulation of pattern recognition recep- Jeong, D. and Green, P. (2013) The role of rice microRNAs in abiotic tor signalling in plants. Nature Reviews Immunology, 16, 537–552. stress responses. Journal of Plant Biology, 56, 187–197. Chen, X. (2009) Small RNAs and their roles in plant development. Annual Jones, J. and Dangl, J. (2006) The plant immune system. Nature, 444, Review of Cell and Developmental Biology, 25, 21–44. 323–329. Chien, P.S., Chiang, C.B., Wang, Z. and Chiou, T.J. (2017) MicroRNA- Lamari, L. (2008) Assess 2.0: Image Analysis Software for Plant Disease mediated signaling and regulation of nutrient transport and utiliza- Quantification. St Paul, MN: APS Press. tion. Current Opinion in Plant Biology, 39, 73–79. Li, Y., Zhang, Q., Zhang, J., Wu, L., Qi, Y. and Zhou, J.M. (2010) Chiou, T.J., Aung, K., Lin, S.I., Wu, C.C., Chiang, S.F. and Su, C.L. (2006) Identification of microRNAs involved in pathogen-associated molec- Regulation of phosphate homeostasis by microRNA in Arabidopsis. ular pattern-triggered plant innate immunity. Plant Physiology, 152, The Plant Cell, 18, 412–421. 2222–2231. CAMPOS-SORIANO et al. | 569 Li, Z.Y., Xia, J., Chen, Z., Yu, Y., Li, Q.F., Zhang, Y.C. et al. (2016) Large Shivaprasad, P.V., Chen, H.M., Patel, K., Bond, D.M., Santos, B.A.C.M. scale rewiring of innate immunity circuitry and microRNA regulation and Baulcombe, D.C. (2012) A MicroRNA superfamily regulates during initial rice blast infection. Scientific Reports, 6, 25493. nucleotide binding site-leucine-rich repeats and other mRNAs. The Liang, G., Ai, Q. and Yu, D. (2015) Uncovering miRNAs involved in cross- Plant Cell, 24, 859–874. talk between nutrient deficiencies in Arabidopsis. Scientific Reports, Song, L. and Liu, D. (2015) Ethylene and plant responses to phosphate 5, 11813. deficiency. Frontiers in Plant Science, 6, 796. Liu, T.Y., Huang, T.K., Tseng, C.Y., Lai, Y.S., Lin, S.I., Lin, W.Y. et al. (2012) Song, X., Li, Y., Cao, X. and Qi, Y. (2019) MicroRNAs and their regula- PHO2-dependent degradation of PHO1 modulates phosphate ho- tory roles in plant–environment interactions. Annual Review of Plant meostasis in Arabidopsis. The Plant Cell, 24, 2168–2183. Biology, 70, 489–525. Llave, C., Xie, Z., Kasschau, K.D. and Carrington, J.C. (2002) Cleavage Supek, F., Bošnjak, M., Škunca, N. and Šmuc, T. (2011) REVIGO summa- of Scarecrow-like mRNA targets directed by a class of Arabidopsis rizes and visualizes long lists of gene ontology terms. PLoS ONE, 6(7), miRNA. Science, 297, 2053–2056. e21800. https​://doi.org/10.1371/journ​al.pone.0021800. Lu, Y.T., Li, M.Y., Cheng, K.T., Tan, C.M., Su, L.W., Lin, W.Y. et al. (2014) Tanaka, K., Choi, J., Cao, Y. and Stacey, G. (2014) Extracellular ATP acts Transgenic plants that express the phytoplasma effector SAP11 as a damage-associated molecular pattern (DAMP) signal in plants. show altered phosphate starvation and defence responses. Plant Frontiers in Plant Science, 5, 446. Physiology, 164, 1456–1469. Tripathi, D., Zhang, T., Koo, A.J., Stacey, G. and Tanaka, K. (2018) Midoh, N. and Iwata, M. (1996) Cloning and characterization of a Extracellular ATP acts on jasmonate signaling to reinforce plant de- probenazole-inducible gene for an intracellular pathogenesis-related fence. Plant Physiology, 176, 511–523. protein in rice. Plant and Cell Physiology, 37(1), 9–18. Veresoglou, S., Barto, E., Menexes, G. and Rillig, M.C. (2013) Fertilization Miedes, E., Vanholme, R., Boerjan, W. and Molina, A. (2014) The role of affects severity of disease caused by fungal plant pathogens. Plant the secondary cell wall in plant resistance to pathogens. Frontiers in Pathology, 62, 961–969. Plant Science, 5, 358. Versaw, W. and Harrison, M.J. (2002) A chloroplast phosphate trans- Misson, J., Raghothama, K.G., Jain, A., Jouhet, J., Block, M.A., Bligny, R. porter, PHT2;1, influences allocation of phosphate within the plant et al. (2005) A genome-wide transcriptional analysis using Arabidopsis and phosphate-starvation responses. The Plant Cell, 14, 1751–1766. thaliana Affymetrix gene chips determined plant responses to phos- Wang, X., Zafian, P., Choudhary, M. and Lawton, M. (1996) The PR5K phate deprivation. Proceedings of the National Academy of Sciences of receptor protein kinase from Arabidopsis thaliana is structurally re- the United States of America, 102, 11934–11939. lated to a family of plant defence proteins. Proceedings of the National Nasir, F., Tian, L., Chang, C., Li, X., Gao, Y., Tran, L.S.P. et al. (2018) Academy of Sciences of the United States of America, 93, 2598–2602. Current understanding of pattern-triggered immunity and hormone- Wang, H., Xu, Q., Kong, Y.H., Chen, Y., Duan, J.Y., Wu, W.H. et al. (2014) mediated defence in rice in response to Magnaporthe oryzae infec- Arabidopsis WRKY45 transcription factor activates PHOSPHATE tion. Seminars in Cell and Developmental Biology, 83, 95–105. TRANSPORTER1;1 expression in response to phosphate starvation. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M. Plant Physiology, 164, 2020–2029. et al. (2006) A plant miRNA contributes to antibacterial resistance by Wasaki, J., Shinano, T., Onishi, K., Yonetani, R., Yazaki, J., Fujii, F. et al. repressing auxin signaling. Science, 312, 436–439. (2006) Transcriptomic analysis indicates putative metabolic changes Oono, Y., Kawahara, Y., Yazawa, T., Kanamori, H., Kuramata, M., caused by manipulation of phosphorus availability in rice leaves. Yamagata, H. et al. (2013) Diversity in the complexity of phosphate Journal of Experimental Botany, 57, 2049–2059. starvation transcriptomes among rice cultivars based on RNA-Seq Wei, T., Ou, B., Li, J., Zhao, Y., Guo, D., Zhu, Y. et al. (2013) Transcriptional profiles. Plant Molecular Biology, 83, 523–537. profiling of rice early response to Magnaporthe oryzae identified Paul, S., Datta, S. and Datta, K. (2015) MiRNA regulation of nutrient OsWRKYs as important regulators in rice blast resistance. PLoS ONE, homeostasis in plants. Frontiers in Plant Science, 6, 232. 8, e59720. Phukan, U.J., Jeena, G.S. and Shukla, R.K. (2016) WRKY Transcription Wilson, R. and Talbot, N. (2009) Under pressure: investigating the bi- factors: molecular regulation and stress responses in plants. Frontiers ology of plant infection by Magnaporthe oryzae. Nature Reviews in Plant Science, 7, 760. Microbiology, 7, 185–195. Puga, M.I., Rojas-Triana, M., de Lorenzo, L., Leyva, A., Rubio, V. and Yamane, H. (2013) Biosynthesis of phytoalexins and regulatory mech- Paz-Ares, J. (2017) Novel insights in the regulation of Pi starvation anisms of it in rice. Bioscience, Biotechnology, and Biochemistry, 77, responses in plants: facts and promises. Current Opinion in Plant 1141–1148. Biology, 39, 40–49. Yang, L., Ding, J., Zhang, C., Jia, J., Weng, H., Liu, W. et al. (2005) Qi, M. and Yang, Y. (2002) Quantification of Magnaporthe grisea during Estimating the copy number of transgenes in transformed rice by infection of rice plants using real-time polymerase chain reaction real-time quantitative PCR. Plant Cell Reports, 23, 759–763. and northern blot/phosphoimaging analyses. Phytopathology, 92, 870–876. Sallaud, C., Meynard, D., van Boxtel, J., Gay, C., Bès, M., Brizard, J.P. S U P P O R T I N G I N FO R M AT I O N et al. (2003) Highly efficient production and characterization Additional Supporting Information may be found online in the of T-DNA plants for rice (Oryza sativa L.) functional genomics. Supporting Information section. Theoretical and Applied Genetics, 106, 1396–1408. Sauter, M. (2015) Phytosulfokine peptide signaling. Journal of Experimental FIGURE S1 Treatment of rice plants (Oryza sativa “Tainung 67”) with Botany, 66, 5161–5169. Secco, D., Jabnoune, M., Walker, H., Shou, H., Wu, P., Poirier, Y. et al. different Pi concentrations. (a) Phenotype of plants that have been (2013) Spatio-temporal transcript profiling of rice roots and shoots grown for 15 days under low-, sufficient-, or high-Pi conditions. (b) in response to phosphate starvation and recovery. The Plant Cell, 25, Shoot fresh weight and dry weight of rice plants grown for 15 days 4285–4304. under different Pi regimes. Data represent means of four biological Shimono, M., Sugano, S., Nakayama, A., Jiang, C.J., Ono, K., Toki, S. et al. (2007) Rice WRKY45 plays a crucial role in benzothiadiazole-induc- replicates, each one from a pool of three different plants, ±SEM (one- ible blast resistance. The Plant Cell, 19, 2064–2076. way analysis of variance and Tukey’s test, *p < .05, **p < .005) 570 | CAMPOS-SORIANO et al. FIGURE S2 Effect of treatment with low- and high-Pi supply in the number of mapping entries). Boxes in the graph show GO terms, susceptible rice cultivars Nipponbare, Maratelli, Bomba, Vialone term definition, and statistical information. The significant terms are Nano, and the resistant cultivar Saber (harbouring the Pib resistance marked with colour, while nonsignificant terms are shown in white gene). Left panels, appearance of plants that have been grown for boxes. The colour intensity of a box positively correlates with the 15 days under different Pi regimes. Right panels, shoot fresh weight enrichment level of the term and dry weight of plants shown in left panels. Data represent means FIGURE S9 GO terms enriched in down-regulated genes in miR399 of four biological replicates, each one from a pool of three different OE plants compared to wild-type plants were generated by AgriGO plants, ±SEM (one-way analysis of variance and Tukey’s test, *p < .05, according to molecular function. Singular enrichment analysis (SEA), **p < .005, ***p < .0001) with the Rice Gramene gene model as a reference, by AgriGO analysis FIGURE S3 Effect of Pi treatment on the expression of Pi starva- (Hypergeometric test; Hochberg FDR; p ≤ .05; “10” as minimum number tion-responsive genes in rice (cv. Tainung 67). The accumulation of of mapping entries). Boxes in the graph show GO terms, term definition, miR399f precursor transcripts and the miR399 target gene OsPHO2 and statistical information. The significant terms are marked with colour, was determined by quantitative reverse transcription PCR. The ex- while nonsignificant terms are shown in white boxes. The colour inten- pression values were normalized to Ubiquitin 1 gene. Bars show the sity of a box positively correlates with the enrichment level of the term means of three biological replicates, with three different plants in FIGURE S10 Heat map visualization of differentially expressed genes in each one, and SEM. Comparisons have been made relative to the suf- miR399 OE plants compared to wild-type (WT) plants in the following ficient Pi condition (t test, *p < .05) subcategories: protein phosphorylation and ATP binding (left panel), FIGURE S4 Characterization of miR399f overexpressing (OE) rice transcriptional regulation (middle panel), and defence (right panel). plants. (a) Accumulation of miR399f precursor and OsPHO2 tran- Expression patterns are based on the expression levels obtained by scripts in miR399 OE lines (T0 generation). (b) Leaf tip necrosis in RNA-Seq (miR399 OE lines L4, L20 versus WT). Purple represents up- leaves of miR399 OE rice plants grown under controlled greenhouse regulated genes (log2 fold change ≥ 0.5) and green represents down- conditions for 2 months regulated genes (log2 fold change ≤ −0.5). Data are means (n = 3). The FIGURE S5 Gene ontology (GO) terms enriched in up-regulated full gene names and locus ID are listed in Tables S3, S4, and S5 genes in miR399OE plants compared to wild-type plants were TABLE S1 Determination of transgene copy number in miR399 generated by AgriGO according to biological process. Singular OE plants by quantitative PCR using sucrose phosphate synthase enrichment analysis (SEA), with the Rice Gramene gene model as gene (OsSPS) as endogenous monocopy reference gene (Ding a reference, by AgriGO analysis (hypergeometic test; Hochberg et al., 2004). Primers sequences are shown in Table S6 (AtmiR399f false discovery rate (FDR); p ≤ .05; “10” as minimum number of and tNOS) mapping entries). Boxes in the graph show GO terms, term defini- TABLE S2 List of up-regulated and down-regulated genes in miR399 tion, and statistical information. The significant terms are marked OE plants compared to wild-type plants. Significantly misregulated with colour, while nonsignificant terms are shown in white boxes. genes with p ≤ .05, FDR ≤ 0.05 (up-regulated, log2 fold change ≥ 0.5; The colour intensity of a box positively correlates with the enrich- down-regulated, log2 fold change ≤ −0.5) ment level of the term TABLE S3 List of genes that are up-regulated and down-regulated in FIGURE S6 GO terms enriched in down-regulated genes in miR399 miR399 OE plants compared to wild-type plants in the category of OE plants compared to wild-type plants were generated by AgriGO protein phosphorylation and ATP binding according to biological process. Singular enrichment analysis (SEA), TABLE S4 List of genes that are up-regulated and down-regulated in with the Rice Gramene gene model as a reference, by AgriGO analy- miR399 OE plants compared to wild-type plants in the category of sis (Hypergeometic test; Hochberg FDR; p ≤ .05; “10” as minimum transcriptional regulation number of mapping entries). Boxes in the graph show GO terms, TABLE S5 List of genes that are up-regulated and down-regulated term definition, and statistical information. The significant terms are in miR399 OE plants compared to wild-type plants in the category marked with colour, while nonsignificant terms are shown in white of defence boxes. The colour intensity of a box positively correlates with the TABLE S6 Sequences of oligonucleotides used for quantitative re- enrichment level of the term verse transcription PCR analysis, northern blot, and transgene copy FIGURE S7 GO terms enriched in miR399 OE plants compared to number determination wild-type plants were generated by AgriGO according to cellular METHODS S1 Experimental procedures component. (a) Functional categories in the group of up-regulated genes. (b) Functional categories in the group of down-regulated genes How to cite this article: Campos-Soriano L, Bundó M, FIGURE S8 GO terms enriched in up-regulated genes in miR399 OE Bach-Pages M, Chiang S-F, Chiou T-J, San Segundo B. plants compared to wild-type plants were generated by AgriGO ac- Phosphate excess increases susceptibility to pathogen cording to molecular function. Singular enrichment analysis (SEA), infection in rice. Molecular Plant Pathology. 2020;21:555–570. with the Rice Gramene gene model as a reference, by AgriGO analy- https​://doi.org/10.1111/mpp.12916​ sis (Hypergeometic test; Hochberg FDR; p ≤ .05; “10” as minimum