Arabidopsis thaliana Early Foliar Proteome Response to Root Exposure to the Rhizobacterium Pseudomonas simiae WCS417.

Pseudomonas simiae WCS417 is a plant growth-promoting rhizobacterium that improves plant health and development. In this study, we investigate the early leaf responses of Arabidopsis thaliana to WCS417 exposure and the possible involvement of formate dehydrogenase (FDH) in such responses. In vitro-grown A. thaliana seedlings expressing an FDH::GUS reporter show a significant increase in FDH promoter activity in their roots and shoots after 7 days of indirect exposure (without contact) to WCS417. After root exposure to WCS417, the leaves of FDH::GUS plants grown in the soil also show an increased FDH promoter activity in hydathodes. To elucidate early foliar responses to WCS417 as well as FDH involvement, the roots of A. thaliana wild-type Col and atfdh1-5 knock-out mutant plants grown in soil were exposed to WCS417, and proteins from rosette leaves were subjected to proteomic analysis. The results reveal that chloroplasts, in particular several components of the photosystems PSI and PSII, as well as members of the glutathione S-transferase family, are among the early targets of the metabolic changes induced by WCS417. Taken together, the alterations in the foliar proteome, as observed in the atfdh1-5 mutant, especially after exposure to WCS417 and involving stress-responsive genes, suggest that FDH is a node in the early events triggered by the interactions between A. thaliana and the rhizobacterium WCS417. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

Cruciferous Weed Isolates of Xanthomonas campestris Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genome-wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection.

Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.

Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.

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.

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

BACKGROUND: The 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). RESULTS: In 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. CONCLUSIONS: This 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.

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.

Bacterial host adaptation through sequence and structural variations of a single type III effector gene.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads.

BACKGROUND: Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. RESULTS: Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. CONCLUSIONS: This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence.

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.

The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis.

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.

Genome Sequences of 17 Strains from Eight Races of Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris is a group of phytopathogenic bacteria causing black rot disease on Brassicaceae crops. Here, we report on draft genome sequences of 17 strains representing eight of nine known races of this pathogen, including the pathotype strain CFBP 6865.

Hydathodes.

Bellenot et al. introduce hydathodes, an oft-overlooked plant organ that acts as a pressure valve to expel excess guttation sap at the leaf margin, typically visible at dawn.

A ß-glucuronidase (GUS) Based Bacterial Competition Assay to Assess Fine Differencesin Fitness during Plant Infection.

Competition assays are a simple phenotyping strategy that confront two bacterial strains to evaluate their relative fitness. Because they are more accurate than single-strain growth assays, competition assays can be used to highlight slight differences that would not otherwise be detectable. In the frame of host-pathogens interactions, they can be very useful to study the contribution of individual bacterial genes to bacterial fitness and lead to the identification of new adaptive traits. Here, we describe how to perform such competition assays by taking the example of the model phytopathogenic bacterium Xanthomonas campestris pv. campestris during infection of the mesophyll of its cauliflower host. This phenotypic assay is based on the use of a Competitive Index (CI) that compares the relative abundance of co-inoculated strains before and after inoculation. Since multiplication is a direct proxy for bacterial fitness, the evolution of the ratio between both strains in the mixed population is a direct way to assess differences in fitness in a given environment. In this protocol, we exploit the blue staining of GUS-expressing bacteria to count blue vs. white colonies on plates and estimate the competitiveness of the strains of interest in plant mesophyll.

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.

SGT1 contributes to coronatine signaling and Pseudomonas syringae pv. tomato disease symptom development in tomato and Arabidopsis.

• Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) causes an economically important bacterial speck disease on tomato and produces symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to a jasmonic acid (JA)-isoleucine analogue, coronatine (COR), produced by Pst DC3000. However, the molecular processes underlying lesion development and COR-induced chlorosis are poorly understood. • In this study, we took advantage of a chlorotic phenotype elicited by COR on Nicotiana benthamiana leaves and virus-induced gene silencing (VIGS) as a rapid reverse genetic screening tool and identified a role for SGT1 (suppressor of G2 allele of skp1) in COR-induced chlorosis. • Silencing of SGT1 in tomato resulted in reduction of disease-associated symptoms (cell death and chlorosis), suggesting a molecular connection between COR-induced chlorosis and cell death. In Arabidopsis, AtSGT1b but not AtSGT1a was required for COR responses, including root growth inhibition and Pst DC3000 symptom (water soaked lesion) development. Notably, overexpression of AtSGT1b did not alter Pst DC3000 symptoms or sensitivity to COR. • Taken together, our results demonstrate that SGT1/SGT1b is required for COR-induced chlorosis and subsequent necrotic disease development in tomato and Arabidopsis. SGT1 is therefore a component of the COR/JA-mediated signal transduction pathway.

Genetic analysis of CAND1-CUL1 interactions in Arabidopsis supports a role for CAND1-mediated cycling of the SCFTIR1 complex.

SKP1-Cullin1-F-box protein (SCF) ubiquitin-ligases regulate numerous aspects of eukaryotic growth and development. Cullin-Associated and Neddylation-Dissociated (CAND1) modulates SCF function through its interactions with the CUL1 subunit. Although biochemical studies with human CAND1 suggested that CAND1 plays a negative regulatory role by sequestering CUL1 and preventing SCF complex assembly, genetic studies in Arabidopsis have shown that cand1 mutants exhibit reduced SCF activity, demonstrating that CAND1 is required for optimal SCF function in vivo. Together, these genetic and biochemical studies have suggested a model of CAND1-mediated cycles of SCF complex assembly and disassembly. Here, using the SCF(TIR1) complex of the Arabidopsis auxin response pathway, we test the SCF cycling model with Arabidopsis mutant derivatives of CAND1 and CUL1 that have opposing effects on the CAND1-CUL1 interaction. We find that the disruption of the CAND1-CUL1 interaction results in an increased abundance of assembled SCF(TIR1) complex. In contrast, stabilization of the CAND1-CUL1 interaction diminishes SCF(TIR1) complex abundance. The fact that both decreased and increased CAND1-CUL1 interactions result in reduced SCF(TIR1) activity in vivo strongly supports the hypothesis that CAND1-mediated cycling is required for optimal SCF function.

Anatomy of leaf apical hydathodes in four monocotyledon plants of economic and academic relevance.

Hydathode is a plant organ responsible for guttation in vascular plants, i.e. the release of droplets at leaf margin or surface. Because this organ connects the plant vasculature to the external environment, it is also a known entry site for several vascular pathogens. In this study, we present a detailed microscopic examination of leaf apical hydathodes in monocots for three crops (maize, rice and sugarcane) and the model plant Brachypodium distachyon. Our study highlights both similarities and specificities of those epithemal hydathodes. These observations will serve as a foundation for future studies on the physiology and the immunity of hydathodes in monocots.