A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity.

Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

Reshaping the Primary Cell Wall: Dual Effects on Plant Resistance to Ralstonia solanacearum and Heat Stress Response.

Temperature elevation drastically affects plant defense responses to Ralstonia solanacearum and inhibits the major source of resistance in Arabidopsis thaliana, mediated by the receptor pair RRS1-R/RPS4. In this study, we refined a previous Genome-Wide Association (GWA) mapping analysis by using a local score approach and detected the primary cell wall CESA3 gene as a major gene involved in plant response to R. solanacearum at both 27°C and elevated temperature, 30°C. We functionally validated CESA3 as a susceptibility gene involved in resistance to R. solanacearum at both 27°C and 30°C through a reverse genetic approach. We provide evidence that the cesa3mre1 mutant enhances resistance to bacterial disease and that resistance is associated with an alteration of root cell morphology conserved at elevated temperature. However, even by forcing the entry of the bacterium to bypass the primary cell wall barrier, the cesa3mre1 mutant still showed enhanced resistance to R. solanacearum with delayed onset of bacterial wilt symptoms. We demonstrated that the cesa3mre1 mutant had constitutive expression of the defense-related gene VSP1 which is up-regulated at elevated temperature and that during infection its expression level is maintained higher than in the wild-type Col-0. In conclusion, this study reveals that alteration of the primary cell wall by mutating the cellulose synthase subunit CESA3 contributes to enhanced resistance to R. solanacearum, remaining effective under heat stress. We expect that these results will help to identify robust genetic sources of resistance to R. solanacearum in the context of global warming.

Network organization of the plant immune system: from pathogen perception to robust defense induction.

The plant immune system has been explored essentially through the study of qualitative resistance, a simple form of immunity, and from a reductionist point of view. The recent identification of genes conferring quantitative disease resistance revealed a large array of functions, suggesting more complex mechanisms. In addition, thanks to the advent of high-throughput analyses and system approaches, our view of the immune system has become more integrative, revealing that plant immunity should rather be seen as a distributed and highly connected molecular network including diverse functions to optimize expression of plant defenses to pathogens. Here, we review the recent progress made to understand the network complexity of regulatory pathways leading to plant immunity, from pathogen perception, through signaling pathways and finally to immune responses. We also analyze the topological organization of these networks and their emergent properties, crucial to predict novel immune functions and test them experimentally. Finally, we report how these networks might be regulated by environmental clues. Although system approaches remain extremely scarce in this area of research, a growing body of evidence indicates that the plant response to combined biotic and abiotic stresses cannot be inferred from responses to individual stresses. A view of possible research avenues in this nascent biology domain is finally proposed.

Tissue-specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology.

Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.

Fight hard or die trying: when plants face pathogens under heat stress.

In their natural environment, plants are exposed to biotic or abiotic stresses that occur sequentially or simultaneously. Plant responses to these stresses have been studied widely and have been well characterised in simplified systems involving single plant species facing individual stress. Temperature elevation is a major abiotic driver of climate change and scenarios have predicted an increase in the number and severity of epidemics. In this context, here we review the available data on the effect of heat stress on plant-pathogen interactions. Considering 45 studies performed on model or crop species, we discuss the possible implications of the optimum growth temperature of plant hosts and pathogens, mode of stress application and temperature variation on resistance modulations. Alarmingly, most identified resistances are altered under temperature elevation, regardless of the plant and pathogen species. Therefore, we have listed current knowledge on heat-dependent plant immune mechanisms and pathogen thermosensory processes, mainly studied in animals and human pathogens, that could help to understand the outcome of plant-pathogen interactions under elevated temperatures. Based on a general overview of the mechanisms involved in plant responses to pathogens, and integrating multiple interactions with the biotic environment, we provide recommendations to optimise plant disease resistance under heat stress and to identify thermotolerant resistance mechanisms.

The Same against Many: AtCML8, a Ca2+ Sensor Acting as a Positive Regulator of Defense Responses against Several Plant Pathogens.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.

Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells, and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared with the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic, as the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple of cell division cycles into those of a shoot, and back, through transdifferentiation.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria.

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.

Immunity at Cauliflower Hydathodes Controls Systemic Infection by Xanthomonas campestris pv campestris.

Hydathodes are water pores found on leaves of a wide range of vascular plants and are the sites of guttation. We report here on the detailed anatomy of cauliflower (Brassicaoleracea) and Arabidopsis (Arabidopsis thaliana) hydathodes. Hydathode surface presents pores resembling stomata giving access to large cavities. Beneath, the epithem is composed of a lacunar and highly vascularized parenchyma offering a direct connection between leaf surface and xylem vessels. Arabidopsis hydathode pores were responsive to ABA and light similar to stomata. The flg22 flagellin peptide, a well-characterized elicitor of plant basal immunity, did not induce closure of hydathode pores in contrast to stomata. Because hydathodes are natural infection routes for several pathogens, we investigated hydathode infection by the adapted vascular phytopathogenic bacterium Xanthomonas campestris pv campestris (Xcc), the causal agent of black rot disease of Brassicaceae. Microscopic observations of hydathodes six days postinoculation indicated a digestion of the epithem cells and a high bacterial multiplication. Postinvasive immunity was shown to limit pathogen growth in the epithem and is actively suppressed by the type III secretion system and its effector proteins. Altogether, these results give a detailed anatomic description of Brassicaceae hydathodes and highlight the efficient use of this tissue as an initial niche for subsequent vascular systemic dissemination of Xcc in distant plant tissues.

Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants.

Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.

Unraveling the early molecular and physiological mechanisms involved in response to phenanthrene exposure.

BACKGROUND: Higher plants have to cope with increasing concentrations of pollutants of both natural and anthropogenic origin. Given their capacity to concentrate and metabolize various compounds including pollutants, plants can be used to treat environmental problems - a process called phytoremediation. However, the molecular mechanisms underlying the stabilization, the extraction, the accumulation and partial or complete degradation of pollutants by plants remain poorly understood. RESULTS: Here, we determined the molecular events involved in the early plant response to phenanthrene, used as a model of polycyclic aromatic hydrocarbons. A transcriptomic and a metabolic analysis strongly suggest that energy availability is the crucial limiting factor leading to high and rapid transcriptional reprogramming that can ultimately lead to death. We show that the accumulation of phenanthrene in leaves inhibits electron transfer and photosynthesis within a few minutes, probably disrupting energy transformation. CONCLUSION: This kinetic analysis improved the resolution of the transcriptome in the initial plant response to phenanthrene, identifying genes that are involved in primary processes set up to sense and detoxify this pollutant but also in molecular mechanisms used by the plant to cope with such harmful stress. The identification of first events involved in plant response to phenanthrene is a key step in the selection of candidates for further functional characterization, with the prospect of engineering efficient ecological detoxification systems for polycyclic aromatic hydrocarbons.

Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids.

After transcription, mRNA editing in angiosperm chloroplasts and mitochondria results in the conversion of cytidine to uridine by deamination. Analysis of Arabidopsis thaliana mutants affected in RNA editing have shown that many pentatricopeptide repeat proteins (PPRs) are required for specific cytidine deamination events. PPR proteins have been shown to be sequence-specific RNA binding proteins allowing the recognition of the C to be edited. The C-terminal DYW domain present in many editing factors has been proposed to catalyze C deamination, as it shows sequence similarities with cytidine deaminases in other organisms. However, many editing factors, such as the first to be discovered, CHLORORESPIRATORY REDUCTION4 (CRR4), lack this domain, so its importance has been unclear. Using a reverse genetic approach, we identified DYW1, an RNA editing factor acting specifically on the plastid ndhD-1 editing site recognized by CRR4. Unlike other known editing factors, DYW1 contains no identifiable PPR motifs but does contain a clear DYW domain. We were able to show interaction between CRR4 and DYW1 by bimolecular fluorescence complementation and to reconstitute a functional chimeric CRR4-DYW1 protein complementing the crr4 dyw1double mutant. We propose that CRR4 and DYW1 act together to edit the ndhD-1 site.

"Omics" Insights into PAH Degradation toward Improved Green Remediation Biotechnologies.

This review summarizes recent knowledge of polycyclic aromatic hydrocarbons (PAHs) biotransformation by microorganisms and plants. Whereas most research has focused on PAH degradation either by plants or microorganisms separately, this review specifically addresses the interactions of plants with their rhizosphere microbial communities. Indeed, plant roots release exudates that contain various nutritional and signaling molecules that influence bacterial and fungal populations. The complex interactions of these populations play a pivotal role in the biodegradation of high-molecular-weight PAHs and other complex molecules. Emerging integrative approaches, such as (meta-) genomics, (meta-) transcriptomics, (meta-) metabolomics, and (meta-) proteomics studies are discussed, emphasizing how "omics" approaches bring new insight into decipher molecular mechanisms of PAH degradation both at the single species and community levels. Such knowledge address new pictures on how organic molecules are cometabolically degraded in a complex ecosystem and should help in setting up novel decontamination strategies based on the rhizosphere interactions between plants and their microbial associates.

Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles.

Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles.

Genome-wide association studies in plant pathosystems: success or failure?

Harnessing natural genetic variation is an established alternative to artificial genetic variation for investigating the molecular dialog between partners in plant pathosystems. Herein, we review the successes of genome-wide association studies (GWAS) in both plants and pathogens. While GWAS in plants confirmed that the genetic architecture of disease resistance is polygenic, dynamic during the infection kinetics, and dependent on the environment, GWAS shortened the time of identification of quantitative trait loci (QTLs) and revealed both complex epistatic networks and a genetic architecture dependent upon the geographical scale. A similar picture emerges from the few GWAS in pathogens. In addition, the ever-increasing number of functionally validated QTLs has revealed new molecular plant defense mechanisms and pathogenicity determinants. Finally, we propose recommendations to better decode the disease triangle.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis.

Quantitative disease resistance (QDR) remains the most prevalent form of plant resistance in crop fields and wild habitats. Genome-wide association studies (GWAS) have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR. To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum, we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R. solanacearum type III effector (T3E) mutants, identified as key pathogenicity determinants after a first screen on an A. thaliana core collection of 25 accessions. Although most quantitative trait loci (QTLs) were highly specific to the identity of the T3E mutant (ripAC, ripAG, ripAQ, and ripU), we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat (NLR) genes that exhibited structural variation. We functionally validated one of these NLRs as a susceptibility factor in response to R. solanacearum, named it Bacterial Wilt Susceptibility 1 (BWS1), and cloned two alleles that conferred contrasting levels of QDR. Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R. solanacearum effectors. In addition, we showed a direct interaction between BWS1 and RipAC T3E, and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b), the latter interaction being suppressed by RipAC. Together, our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC, mediating negative regulation of the SGT1-dependent immune response.

The ecologically relevant genetics of plant-plant interactions.

Interactions among plants have been long recognized as a major force driving plant community dynamics and crop yield. Surprisingly, our knowledge of the ecological genetics associated with variation of plant-plant interactions remains limited. In this opinion article by scientists from complementary disciplines, the international PLANTCOM network identified four timely questions to foster a better understanding of the mechanisms mediating plant assemblages. We propose that by identifying the key relationships among phenotypic traits involved in plant-plant interactions and the underlying adaptive genetic and molecular pathways, while considering environmental fluctuations at diverse spatial and time scales, we can improve predictions of genotype-by-genotype-by-environment interactions and modeling of productive and stable plant assemblages in wild habitats and crop fields.

Arabidopsis roots and shoots show distinct temporal adaptation patterns toward nitrogen starvation.

Nitrogen (N) is an essential macronutrient for plants. N levels in soil vary widely, and plants have developed strategies to cope with N deficiency. However, the regulation of these adaptive responses and the coordinating signals that underlie them are still poorly understood. The aim of this study was to characterize N starvation in adult Arabidopsis (Arabidopsis thaliana) plants in a spatiotemporal manner by an integrative, multilevel global approach analyzing growth, metabolites, enzyme activities, and transcript levels. We determined that the remobilization of N and carbon compounds to the growing roots occurred long before the internal N stores became depleted. A global metabolite analysis by gas chromatography-mass spectrometry revealed organ-specific differences in the metabolic adaptation to complete N starvation, for example, for several tricarboxylic acid cycle intermediates, but also for carbohydrates, secondary products, and phosphate. The activities of central N metabolism enzymes and the capacity for nitrate uptake adapted to N starvation by favoring N remobilization and by increasing the high-affinity nitrate uptake capacity after long-term starvation. Changes in the transcriptome confirmed earlier studies and added a new dimension by revealing specific spatiotemporal patterns and several unknown N starvation-regulated genes, including new predicted small RNA genes. No global correlation between metabolites, enzyme activities, and transcripts was evident. However, this multilevel spatiotemporal global study revealed numerous new patterns of adaptation mechanisms to N starvation. In the context of a sustainable agriculture, this work will give new insight for the production of crops with increased N use efficiency.

Study of natural diversity in response to a key pathogenicity regulator of Ralstonia solanacearum reveals new susceptibility genes in Arabidopsis thaliana.

Ralstonia solanacearum gram-negative phytopathogenic bacterium exerts its virulence through a type III secretion system (T3SS) that translocates type III effectors (T3Es) directly into the host cells. T3E secretion is finely controlled at the posttranslational level by helper proteins, T3SS control proteins, and type III chaperones. The HpaP protein, one of the type III secretion substrate specificity switch (T3S4) proteins, was previously highlighted as a virulence factor on Arabidopsis thaliana Col-0 accession. In this study, we set up a genome-wide association analysis to explore the natural diversity of response to the hpaP mutant of two A. thaliana mapping populations: a worldwide collection and a local population. Quantitative genetic variation revealed different genetic architectures in both mapping populations, with a global delayed response to the hpaP mutant compared to the GMI1000 wild-type strain. We have identified several quantitative trait loci (QTLs) associated with the hpaP mutant inoculation. The genes underlying these QTLs are involved in different and specific biological processes, some of which were demonstrated important for R. solanacearum virulence. We focused our study on four candidate genes, RKL1, IRE3, RACK1B, and PEX3, identified using the worldwide collection, and validated three of them as susceptibility factors. Our findings demonstrate that the study of the natural diversity of plant response to a R. solanacearum mutant in a key regulator of virulence is an original and powerful strategy to identify genes directly or indirectly targeted by the pathogen.

Xanthomonas transcriptome inside cauliflower hydathodes reveals bacterial virulence strategies and physiological adaptations at early infection stages.

Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes, which are organs localized at leaf margins. To decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analysed 3 days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and despite a biotrophic lifestyle, 18% of Xcc genes were differentially expressed, including a striking repression of chemotaxis and motility functions. The Xcc full repertoire of virulence factors had not yet been activated but the expression of the HrpG regulon composed of 95 genes, including genes coding for the type III secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulphur and phosphate metabolism was upregulated while limited stress responses were observed 3 days postinfection. We confirmed experimentally that high-affinity phosphate transport is needed for bacterial fitness inside hydathodes. This analysis provides information about the nutritional and stress status of bacteria during the early biotrophic infection stages and helps to decipher the adaptive strategy of Xcc to the hydathode environment.