Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

Response of Xanthomonas citri pv. malvacearum isolates to cotton differing in susceptibility to the bacterium and their predicted type III effectors.

From 2015 to 2020, 342 isolates of Xanthomonas citri pv. malvacearum (Xcm) were obtained from cotton fields in Texas, including 64 isolates collected from symptomatic cultivars that were thought to be resistant to race 18 strains of Xcm (the predominant race in the USA). Symptoms on highly resistant cultivars prompted concern that a new race of Xcm was present. The 342 isolates were inoculated on a race 18 susceptible (DP 1747NR B2XF) and race 18 resistant (S295) cotton cultivar and none of the isolates caused blight type symptoms (water soaking and chlorosis) on S295, indicating that the B12 resistant gene was still beneficial for disease management. Four cultivars, varying in their field response to bacterial blight, were inoculated with each of 17 isolates of Xcm and the incidence of plants exhibiting bacterial blight symptoms averaged 87% for DP 1747NR B2XF, 51% for partially susceptible NG 4936 B3XF, 16% for partially resistant DP 1646 B2XF, and 0% for S295. Xcm isolates from Texas (11), Georgia (1) and Oklahoma (1) were sequenced, and their type three effectors (T3Es) were predicted. All isolates (GA, OK, TX) had the same T3E proteins as previously identified Xcm race 18 isolates (tested for 25 genes), including XopJ. Race 1, 2, 3, and 12 of Xcm included in the comparisons were all missing the XopJ gene. Use of cultivars with the B12 gene is an effective strategy to manage bacterial blight of cotton.

An NLR Integrated Domain toolkit to identify plant pathogen effector targets

SUMMARYPlant resistance genes (or NLR “Nod-like Receptors”) are known to contain atypical domains procuring them with a decoy capacity. Some of these integrated domains (or ID) allow the plant to lure the virulence determinants (“effectors”) of pathogens and triggering a specific NLR immune reaction.In this work, our goal was to generate a library of known IDs that could be screened with plant pathogen effectors in order to identify putative new effector virulence targets and NLR-effector pairs.We curated the IDs contained in NLRs from seven model and crop plant species. We cloned 52 IDs representing 31 distinct Pfam domains. This library was screened for interaction by yeast-two-hybrid with a set of 31 conserved Ralstonia solanacearum type III effectors. This screening and the further in planta interaction assay allowed us to identify three interactions, involving different IDs (kinase, DUF3542, WRKY) and two type III effectors (RipAE and PopP2).PopP2 was found to physically interact with ID#85, an atypical WRKY domain integrated in the GmNLR-ID85 NLR protein from Soybean. Using a imaging method in living plant cells, we showed that PopP2 associates with ID#85 in the nucleus. But unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, this newly identified soybean WRKY domain could not be acetylated by PopP2 and its atypical sequence (WRKYGKR) also probably renders it inefficient in plant immunity triggering.This ID toolkit is available for screening with other plant pathogen effectors and should prove useful to discover new effectors targets and potentially engineer new plant resistance genes.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Xanthomonas effector XopR hijacks host actin cytoskeleton via complex coacervation

The intrinsically disordered region (IDR) is a preserved signature of phytobacterial type III effectors (T3Es). The T3E IDR is thought to mediate unfolding during translocation into the host cell and to avoid host defense by sequence diversification. Here, we demonstrate a mechanism of host subversion via the T3E IDR. We report that the Xanthomonas campestris T3E XopR undergoes liquid-liquid phase separation (LLPS) via multivalent IDR-mediated interactions that hijack the Arabidopsis actin cytoskeleton. XopR is gradually translocated into host cells during infection and forms a macromolecular complex with actin-binding proteins at the cell cortex. By tuning the physical-chemical properties of XopR-complex coacervates, XopR progressively manipulates multiple steps of actin assembly, including formin-mediated nucleation, crosslinking of F-actin, and actin depolymerization, which occurs through competition for actin-depolymerizing factor and depends on constituent stoichiometry. Our findings unravel a sophisticated strategy in which bacterial T3E subverts the host actin cytoskeleton via protein complex coacervation.

Publisher Correction: Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species

The Bradyrhizobium vignae strain ORS3257 is an elite strain recommended for cowpea inoculation in Senegal. This strain was recently shown to establish symbioses on some Aeschynomene species using a cocktail of Type III effectors (T3Es) secreted by the T3SS machinery. In this study, using a collection of mutants in different T3Es genes, we sought to identify the effectors that modulate the symbiotic properties of ORS3257 in three Vigna species (V. unguiculata, V. radiata and V. mungo). While the T3SS had a positive impact on the symbiotic efficiency of the strain in V. unguiculata and V. mungo, it blocked symbiosis with V. radiata. The combination of effectors promoting nodulation in V. unguiculata and V. mungo differed, in both cases, NopT and NopAB were involved, suggesting they are key determinants for nodulation, and to a lesser extent, NopM1 and NopP1, which are additionally required for optimal symbiosis with V. mungo. In contrast, only one effector, NopP2, was identified as the cause of the incompatibility between ORS3257 and V. radiata. The identification of key effectors which promote symbiotic efficiency or render the interaction incompatible is important for the development of inoculation strategies to improve the growth of Vigna species cultivated in Africa and Asia.

Proteomic analysis of potato responding to the invasion of Ralstonia solanacearum UW551 and its type III secretion system mutant

The infection of potato with Ralstonia solanacearum (R. solanacearum) UW551 gives rise to bacterial wilt disease via colonization of roots. The Type III Secretion System (T3SS) is a determinant factor for the pathogenicity of R. solanacearum. To fully understand perturbations in potato by R. solanacearum Type III Effectors(T3Es), we use proteomics to measure differences in potato root protein abundance after inoculation with R. solanacearum UW551 and T3SS mutant (UW551△HrcV). We identified 21 differentially accumulated proteins (DAPs). Compared to inoculation with UW551△HrcV, ten proteins showed significantly lower abundance in potato roots after inoculation with UW551, indicating those proteins were significantly down-regulated by T3Es during the invasion. To identify their functions in immunity, we silenced those genes in Nicotiana benthamiana and tested the resistance of the silenced plants to the pathogen. Results showed that miraculin, HBP2, TOM20 contribute to immunity to R. solanacearum. In contrast, PP1 contribute to susceptibility. Notably, none of four downregulated proteins (HBP2, PP1, HSP22, TOM20) were downregulated at the transcriptional level, suggesting that they were significantly down-regulated at the post-transcriptional level. We further co-expressed those four proteins with thirty-three core T3Es. To our surprise, multiple effectors were able to significantly decrease the sudied protein abundances. In conclusion, our data showed T3Es of R. solanacearum could subvert potato root immune-related proteins in a redundant manner.