Nicolas Denancé

Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs

Plant growth and response to environmental cues are largely governed by phytohormones. The plant hormones ethylene, jasmonic acid, and salicylic acid (SA) play a central role in the regulation of plant immune responses. In addition, other plant hormones, such as auxins, abscisic acid (ABA), cytokinins, gibberellins, and brassinosteroids, that have been thoroughly described to regulate plant development and growth, have recently emerged as key regulators of plant immunity. Plant hormones interact in complex networks to balance the response to developmental and environmental cues and thus limiting defense-associated fitness costs. The molecular mechanisms that govern these hormonal networks are largely unknown. Moreover, hormone signaling pathways are targeted by pathogens to disturb and evade plant defense responses. In this review, we address novel insights on the regulatory roles of the ABA, SA, and auxin in plant resistance to pathogens and we describe the complex interactions among their signal transduction pathways. The strategies developed by pathogens to evade hormone-mediated defensive responses are also described. Based on these data we discuss how hormone signaling could be manipulated to improve the resistance of crops to pathogens.

Walls are thin 1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers.

By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. Walls are thin 1 (WAT1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN 21 (MtN21), encodes a plant-specific, predicted integral membrane protein, and is a member of the plant drug/metabolite exporter (P-DME) family (transporter classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues, including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized to the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation, resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild-type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers.

Post mortem function of AtMC9 in xylem vessel elements

Cell death of xylem elements is manifested by rupture of the tonoplast and subsequent autolysis of the cellular contents. Metacaspases have been implicated in various forms of plant cell death but regulation and execution of xylem cell death by metacaspases remains unknown. Analysis of the type II metacaspase gene family in Arabidopsis thaliana supported the function of METACASPASE 9 (AtMC9) in xylem cell death. Progression of xylem cell death was analysed in protoxylem vessel elements of 3-d-old atmc9 mutant roots using reporter gene analysis and electron microscopy. Protoxylem cell death was normally initiated in atmc9 mutant lines, but detailed electron microscopic analyses revealed a role for AtMC9 in clearance of the cell contents post mortem, that is after tonoplast rupture. Subcellular localization of fluorescent AtMC9 reporter fusions supported a post mortem role for AtMC9. Further, probe-based activity profiling suggested a function of AtMC9 on activities of papain-like cysteine proteases. Our data demonstrate that the function of AtMC9 in xylem cell death is to degrade vessel cell contents after vacuolar rupture. We further provide evidence on a proteolytic cascade in post mortem autolysis of xylem vessel elements and suggest that AtMC9 is part of this cascade.

Emerging Functions of Nodulin-Like Proteins in Non-Nodulating Plant Species

Plant genes whose expression is induced in legumes by Rhizobium bacteria upon nodulation were initially referred to as nodulins. Several of them play a key role in the establishment of symbiosis. Yet, nodulin-like proteins are also found in non-nodulating plant species such as Arabidopsis, rice, maize or poplar. For instance, 132 are predicted in the Arabidopsis thaliana Col-0 genome. Recent studies now highlight the importance of nodulin-like proteins for the transport of nutrients, solutes, amino acids or hormones and for major aspects of plant development. Interestingly, nodulin-like activities at the plant-microbe interface are also important for pathogens to enhance their fitness during host colonization. This work presents a genomic and functional overview of nodulin-like proteins in non-leguminous plant species, with a particular focus on Arabidopsis and rice.

Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism

Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas†

How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome

BackgroundThe bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani).ResultsIn this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins.ConclusionsThis dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.

PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis

PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R. solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

Predicting promoters targeted by TAL effectors in plant genomes: from dream to reality

Transcription Activator-Like (TAL) effectors from the plant pathogenic bacteria of the genus Xanthomonas are molecular weapons injected into eukaryotic cells to modulate the host transcriptome. Upon delivery, TAL effectors localize into the host cell nucleus and bind to the promoter of plant susceptibility (S) genes to activate their expression and thereby facilitate bacterial multiplication (Boch and Bonas, 2010; Schornack et al., 2013). In resistant plants, a few TAL effectors have been shown to bind to promoters of executor resistance (R) genes, resulting in localized cell death and preventing pathogen spread (reviewed in Doyle et al., 2013). Remarkably, TAL effectors harbor a novel type of DNA-binding domain with a unique modular architecture composed of 1.5–33.5 almost identical tandem repeats of 33–35 amino acids. Each repeat type specifies one or more bases through direct interaction with the second amino acid in a centrally located “Repeat Variable Diresidue” (RVD). The number and sequence of the RVDs across the whole repeat region of the TAL protein defines the DNA target. The code of DNA-binding specificity of Xanthomonas TAL effectors was inferred from experimental, computational and later on structural approaches (Boch et al., 2009; Moscou and Bogdanove, 2009; Deng et al., 2012; Mak et al., 2012). This new paradigm for protein-DNA interaction is now revolutionizing our perspectives for the understanding of TAL effectors roles during plant disease and defense since the identification of their plant targets is largely facilitated. A few algorithms are now available to predict in silico candidate genes of a given TAL effector. This Opinion gives an overview of the current tools and strategies that may be applied for finding targets of TAL effectors. We also raise limitations and pitfalls and emphasize what may be improved to gain in prediction accuracy. Finally, we also highlight several perspectives offered by these new tools.

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination

ABSTRACT Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants.