Rhizobacterium‐derived diacetyl modulates plant immunity in a phosphate‐dependent manner

Article5 December 2019Open Access Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner Rafael JL Morcillo Rafael JL Morcillo Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Sunil K Singh Sunil K Singh Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Danxia He Danxia He Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guo An Guo An Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Juan I Vílchez Juan I Vílchez Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Kai Tang Kai Tang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Fengtong Yuan Fengtong Yuan Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yazhou Sun Yazhou Sun Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chuyang Shao Chuyang Shao Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Song Zhang Song Zhang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yu Yang Yu Yang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xiaomin Liu Xiaomin Liu Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yashan Dang Yashan Dang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wei Wang Wei Wang Shanghai Chenshan Botanical Garden, Shanghai, China Search for more papers by this author Jinghui Gao Jinghui Gao College of Grassland Agriculture, Northwest A&F University, Yangling, China Search for more papers by this author Weichang Huang Weichang Huang Shanghai Chenshan Botanical Garden, Shanghai, China Search for more papers by this author Mingguang Lei Mingguang Lei Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Chun-Peng Song Chun-Peng Song State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, Kaifeng, China Search for more papers by this author Jian-Kang Zhu Jian-Kang Zhu Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Alberto P Macho Alberto P Macho Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Pual W Paré Pual W Paré Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, TX, USA Search for more papers by this author Huiming Zhang Corresponding Author Huiming Zhang [email protected] orcid.org/0000-0003-0695-3593 Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, Kaifeng, China Search for more papers by this author Rafael JL Morcillo Rafael JL Morcillo Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Sunil K Singh Sunil K Singh Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Danxia He Danxia He Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Guo An Guo An Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Juan I Vílchez Juan I Vílchez Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Kai Tang Kai Tang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Fengtong Yuan Fengtong Yuan Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yazhou Sun Yazhou Sun Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chuyang Shao Chuyang Shao Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Song Zhang Song Zhang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yu Yang Yu Yang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xiaomin Liu Xiaomin Liu Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yashan Dang Yashan Dang Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Wei Wang Wei Wang Shanghai Chenshan Botanical Garden, Shanghai, China Search for more papers by this author Jinghui Gao Jinghui Gao College of Grassland Agriculture, Northwest A&F University, Yangling, China Search for more papers by this author Weichang Huang Weichang Huang Shanghai Chenshan Botanical Garden, Shanghai, China Search for more papers by this author Mingguang Lei Mingguang Lei Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Chun-Peng Song Chun-Peng Song State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, Kaifeng, China Search for more papers by this author Jian-Kang Zhu Jian-Kang Zhu Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Department of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN, USA Search for more papers by this author Alberto P Macho Alberto P Macho Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Pual W Paré Pual W Paré Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, TX, USA Search for more papers by this author Huiming Zhang Corresponding Author Huiming Zhang [email protected] orcid.org/0000-0003-0695-3593 Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, Kaifeng, China Search for more papers by this author Author Information Rafael JL Morcillo1,‡, Sunil K Singh1,‡, Danxia He1,2,‡, Guo An1,2,‡, Juan I Vílchez1, Kai Tang1,3, Fengtong Yuan1,2, Yazhou Sun1,2, Chuyang Shao1,2, Song Zhang1, Yu Yang1, Xiaomin Liu1,2, Yashan Dang1,2, Wei Wang4, Jinghui Gao5, Weichang Huang4, Mingguang Lei1, Chun-Peng Song6, Jian-Kang Zhu1,3, Alberto P Macho1, Pual W Paré7 and Huiming Zhang *,1,6 1Shanghai Center for Plant Stress Biology, and CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China 2University of Chinese Academy of Sciences, Beijing, China 3Department of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN, USA 4Shanghai Chenshan Botanical Garden, Shanghai, China 5College of Grassland Agriculture, Northwest A&F University, Yangling, China 6State Key Laboratory of Crop Stress Adaptation and Improvement, Henan University, Kaifeng, China 7Department of Chemistry & Biochemistry, Texas Tech University, Lubbock, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 2157 078248; E-mail: [email protected] The EMBO Journal (2020)39:e102602https://doi.org/10.15252/embj.2019102602 See also: A Zuccaro (January 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity. Synopsis A volatile compound produced by a plant growth-promoting rhizobacterium differentially affects phytohormone-mediated immunity in phosphate-sufficient and -deficient plants, thus modulating their response to symbiont colonization. Exposure to a plant growth-promoting rhizobacterium Bacillus amyloliquefaciens can cause either beneficial or deleterious effects on Arabidopsis thaliana plants depending on their nutrition status. The beneficial vs. deleterious effects of the rhizobacterium depend on the availability of phosphate (Pi) to the plant. The effect of the rhizobacterium on plant fitness is mediated by the volatile bacterial metabolite diacetyl (DA). In Pi-sufficient plants, DA partially suppresses plant immune response and enhances symbiont colonization without compromising disease resistance. In Pi-deficient plants, DA enhances phytohormone-mediated immune response and causes plant hypersensitivity to Pi deficiency. Introduction Plants naturally live with a diversity of microbes in the rhizosphere and the phyllosphere. Some microbes are considered as beneficial due to their capacities of increasing plant biomass and/or stress tolerance (Lugtenberg & Kamilova, 2009; Pieterse et al, 2014). In return, plants provide nutrient-rich root exudates and niches for beneficial microbes, leading to mutualistic symbiosis (Sasse et al, 2008). Plants activate immune responses including production of reactive oxygen species (ROS) to combat pathogens (Yan & Dong, 2014; Couto & Zipfel, 2016; Wu et al, 2018). When encountering beneficial microbes, a balanced regulation of plant immunity is presumably required for mutualistic associations. Plant immune responses can be triggered by pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin; similarly, establishment of mutualistic symbiosis involves plant perception of microbial symbiotic signals, as demonstrated by symbiosis with arbuscular mycorrhizal fungi or nodule-forming rhizobia (Cao et al, 2017; Martin et al, 2017; Zipfel & Oldroyd, 2017). Nonetheless, the interplay between mutualism and immunity is unclear. The establishment of Arabidopsis symbiosis with Colletotrichum tofieldiae, an endophytic fungus that can transfer phosphate to its host, requires phosphate (Pi) deficiency in the plant (Hacquard et al, 2016; Hiruma et al, 2016). In addition, the master regulators of phosphate-starvation response (PSR) in Arabidopsis not only positively regulate PSR but also suppress plant immunity, and thereby influence root microbiome (Castrillo et al, 2017). It is thus intriguing whether plant mutualistic associations with beneficial soil microbes commonly prefer Pi deficiency. Bacillus amyloliquefaciens strain GB03 and its microbial volatiles (hereafter referred to as GMVs) are recognized as beneficial to plants both in soil and in artificial medium. GMVs were shown to modulate plant hormone homeostasis and nutrient uptake (Ryu et al, 2003; Zhang et al, 2007, 2009; Paré et al, 2011; Beauregard et al, 2013); however, it remains unclear how plant biological processes were integrated by the microbial factors to produce multiple beneficial traits (Paré et al, 2011; Liu & Zhang, 2015). In this study, we show that Arabidopsis allows mutualistic association with B. amyloliquefaciens GB03 only under the Pi-sufficient condition, whereas Pi-deficient plants strongly activate immunity in response to the same bacterium. Our investigation further identified a bacterial volatile compound that influences the plant decision on mutualism or immunity. Our findings not only demonstrate that bacterial factor-triggered modulation of the immune system and the PSR system in plants determines the relationship between the two organisms, but also provide an example where plants use different strategies for bacteria and fungi in determining mutualism or immunity. Results A plant abiotic stress condition disclosed a mutualism-to-pathogenicity transition We were initially interested in studying whether GB03 would relieve plant stress caused by simultaneous deficiency of multiple nutrients. In order to do this, we grew seedlings of Arabidopsis thaliana in petri dishes containing 1/2-strength and 1/20-strength Murashige and Skoog medium as the nutrient-sufficient and nutrient-deficient medium, respectively. The petri dishes contained plastic partitions which separated different medium and also separated plants from the bacteria, so that the bacteria could influence plants only through volatile emissions (Fig 1A). In such conditions, we unexpectedly observed deleterious effects of GMVs on Arabidopsis grown in nutrient-deficient medium, while the same GMVs promoted growth of plants supplemented with sufficient nutrients (Figs 1A and EV1A). Under nutrient-deficient conditions, Arabidopsis not only lose GMV-induced plant growth promotion (Figs 1B and EV1B), but also clearly displayed stress symptoms, including impaired photosynthesis (Fig 1C), increased leaf cell death (Fig 1D), strong accumulation of anthocyanin (Fig EV1C), and hyper-induction or reduction of genes known to be up- or down-regulated, respectively, by environmental stress (Fig EV1D). Thus, GMVs can be either beneficial or deleterious to plants, although GB03 has been recognized as a representative plant mutualistic bacterium (Paré et al, 2011; Choi et al, 2014). Figure 1. A plant abiotic stress condition disclosed a mutualism-to-pathogenicity transition A. The same MVs from GB03 caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. 0.5 MS and 0.05 MS indicate 1/2-strength (nutrient-sufficient) and 1/20-strength (nutrient-deficient) Murashige and Skoog medium. Images were taken at 11 days after treatments (DAT). B. Quantification of total leaf area per plant (square centimeter, cm2). Values correspond to the means ± SE of three biological replicates. Different letters denote significant differences at P < 0.05, Tukey's multiple comparison test within each group of the same DAT. C. Effective quantum yield of photosystem II in plants. n = 3 biological replicates, mean ± SE. Asterisks denote significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001, Dunnett's multiple comparison test (comparing with the control) within each group of the same DAT D. Cell-death visualization by trypan blue staining of 11 DAT leaves. Scale bar = 1 mm. E. Gene Ontology (GO) comparative analysis of Arabidopsis genes that were induced at 5 DAT by nutrient deficiency alone (0.05C vs. 0.5C) and that were induced by the nutrient deficiency plus GMVs (0.05T vs. 0.5C). Diagrams are designed based on VirtualPlant platform. The size of circles represents the number of genes in each GO category. Scale color bar indicates the P-value cutoff of over-representation equal or less than the cutoff for each GO category. Darker color indicates higher possibility of each GO category. DEG lists for key terms are provided in Tables EV1 and EV2. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Nutrient availability determines Arabidopsis thaliana responses to Bacillus amyloliquefaciens GB03 (related to Fig 1) A. Exposure to MVs from GB03, which was either grown on the nutrient-sufficient (0.5 MS) or grown on the nutrient-deficient (0.05 MS) medium, caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. B. Quantification of fresh weight of seedlings grown under different treatment conditions. Values correspond to the means ± SE of three biological replicates. Different letters denote significant differences at P < 0.05, Tukey's multiple comparison test within each group of the same DAT. C. Anthocyanin accumulation levels in plants at 11 DAT. The boxplots show representative data from three independent experiments (n = 9). Whiskers represent the min to max data range, and the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartiles. Different letters denote significantly different means at P < 0.05, Tukey's multiple comparison test. D. Relative gene expression levels of Arabidopsis ELI3, MYB75, MDAR3, and GRXC11, which are indicative of environmental stress conditions (Somssich et al, 1996; Teng et al, 2005; Li et al, 2010; Mehterova et al, 2012), in 5 DAT seedlings grown in 0.5 and 0.05 MS medium with or without exposure to GMVs. Values are means ± SE of three biological replicates. Different letters denote significantly different means at P < 0.05, Tukey's multiple comparison test. E. Gene Ontology (GO) comparative analysis of Arabidopsis genes that were repressed at 5 DAT by nutrient deficiency (0.05C vs. 0.5C) alone and that were repressed by the nutrient deficiency plus GMVs (0.05T vs. 0.5C). Diagrams are designed based on VirtualPlant platform. The size of circles represents the number of genes in each GO category. Scale color bar indicates the P-value cutoff of over-representation equal or less than the cutoff for each GO category. Darker color indicates higher possibility of each GO category. DEG lists for key terms are provided in Table EV3. Download figure Download PowerPoint To understand how GMVs exacerbated the stress in plants that are under nutrient deficiency, we compared the transcriptomes of Arabidopsis with and without GMV treatment under nutrient-deficient conditions. Gene Ontology (GO) analysis of RNAseq results revealed that genes induced by nutrient deficiency were enriched in immune response and phosphate metabolic response processes, and that these patterns were strongly intensified by GMVs (Fig 1E; Appendix Fig S1C; Tables EV1 and EV4). Compared with nutrient deficiency alone, nutrient deficiency with GMV treatment also additionally induced cell-death genes in plants (Fig 1E; Table EV2). These results indicate that GMV-induced stress in Arabidopsis is mediated through microbial regulation of plant immunity and phosphate homeostasis. Meanwhile, genes that were repressed by nutrient deficiency were enriched in hormone response processes, among which genes responsive to gibberellic acid (GA) were repressed only in nutrient-deficient plants with GMV treatment (Fig EV1E; Table EV3), suggesting that GMVs inhibit GA-mediated plant growth. Under nutrient-sufficient conditions, GMVs induced genes related to cell wall organization and photosynthesis (Appendix Fig S1A; Table EV4), consistent with previous reports that GMVs induced leaf cell expansion and enhanced photosynthesis efficiency of Arabidopsis grown in artificial medium (Zhang et al, 2007, 2008). Interestingly, in contrast to GMV-activated immunity in nutrient-deficient plants, GMV treatment to nutrient-sufficient plants repressed immunity-related genes involved in plant responses to fungus, bacteria, chitin, jasmonic acid (JA), and salicylic acid (SA) (Appendix Fig S1B; Table EV4). In nutrient-deficient condition, GMVs induced phosphate starvation, jasmonic acid signaling, and responses triggered by lipid metabolism (Appendix Fig S1C; Table EV4) and repress responses to reactive oxygen species and primary root development (Appendix Fig S1D; Table EV4). Altogether, the transcriptome results suggest that plant immunity and Pi homeostasis are strongly correlated with GMV-induced plant vigor or stress. Phosphate availability determines Arabidopsis responses to GMVs Because Pi homeostasis was highlighted in the transcriptional analysis of nutrient-deficient plants, we wondered whether GMVs affect the plant PSR. As shown in the RNAseq results, while nutrient deficiency induced most of the Pi homeostasis genes in Arabidopsis, these genes were further induced by GMVs (Fig EV2A; Table EV5), demonstrating that GMVs caused plant hyper-activation of the PSR. This conclusion was confirmed by organ-specific measurements of PSR gene expression, which showed that PSR gene hyper-induction in both shoots and roots was generally observable starting at 5 days after treatments (Figs 2A, and EV2B and C). GMV-induced hyper-PSR was further supported by the observations that, in nutrient-deficient plants, GMVs increased the activity of root acid phosphatases and the accumulation of the microRNA miR399 (Figs 2B and EV2D), which are also known to be induced by Pi deficiency in Arabidopsis (Fujii et al, 2005; Zhang et al, 2014b). Therefore, exposure to GMVs resulted in hyper-stimulated PSR in the nutrient-deficient plants. Click here to expand this figure. Figure EV2. Phosphate availability determines Arabidopsis responses to GMVs (related to Fig 2) A. A heatmap of RNAseq results showing the expression levels of Pi homeostasis genes in Arabidopsis grown under different conditions, including 0.5T (0.5 MS medium with GMV treatment), 0.05T (0.05 MS medium with GMV treatment), and 0.05C (0.05 MS medium without GMV treatment). Color scale indicates fold changes (log2) compared with gene expression in plants grown in 0.5C (0.5 MS medium without GMV treatment). DEG lists are provided in Table EV5. B, C. GMVs caused plant hyper-sensitivity to Pi deficiency, as shown by GMV-dependent hyper-induction of PHT1.7 (B) and hyper-suppression of PHO2. (C) Data points indicate mean ± SE (n = 3). Different letters denote significantly different means at P < 0.05, Tukey's multiple comparison test within each group of the same DAT. D. The accumulation level of miR399 was strongly elevated by GMVs in nutrient-deficient plants. Values are means ± SE of three biological replicates. Different letters denote significantly different means at P < 0.05, Tukey's multiple comparison test within each group of the same DAT. E. Different macronutrients including phosphorus (P), nitrogen (N), potassium (K), calcium (Ca), and sulfur (S) were supplemented individually to the 0.05 MS medium, in order to bring the corresponding nutrient content to a level that is equal to that in the 0.5 MS medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. A scheme showing medium partitions is shown on the top right corner. Download figure Download PowerPoint Figure 2. Phosphate availability determines Arabidopsis responses to GMVs A. Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates. B. Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots. C, D. Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) and hyper-induction of IPS1 gene expression (D) triggered by GMVs. E. Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. F. A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. G. The Arabidopsis phr1phl1 mutant showed substantially decreased growth promotion, compared with the wild-type plants. Data information: The boxplots show representative data from three independent experiments (n = 12). Whiskers represent the min to max data range, and the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartiles. Different letters denote significantly different means at P < 0.05, Tukey's multiple comparison test within each group of the same DAT. Download figure Download PowerPoint We next tested whether Pi was the main nutrient that determined the transition between GMV-triggered plant vigor and stress in conditions of nutrient shortage. Phosphate supplementation to nutrient-deficient plants blocked GMV-dependent hyper-activation of PSR genes and substantially reduced anthocyanin accumulation (Figs 2C and D, and EV2E), indicating that GMV-induced stress in the nutrient-deficient plants mainly resulted from Pi deficiency. In contrast, supplementation of several other nutrients including nitrogen (N), potassium (K), sulfur (S), and calcium (Ca) to the nutrient-deficient medium did not decrease GMV-induced stress symptoms in plants (Fig EV2E). Thus, in the medium with simultaneous deficiency of multiple nutrients, Pi deficiency is the major reason for GMV-induced plant stress. Consistently, when plants were grown in the medium that was only deficient in Pi but not in the other nutrients, th