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

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). 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. 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

The Arabidopsis RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) is a biotic stress susceptibility gene

© 2018, The Author(s). Crop breeding for improved disease resistance may be achieved through the manipulation of host susceptibility genes. Previously we identified multiple Arabidopsis mutants known as enhanced stress response1 (esr1) that have defects in a KH-domain RNA-binding protein and conferred increased resistance to the root fungal pathogen Fusarium oxysporum. Here, screening the same mutagenized population we discovered two further enhanced stress response mutants that also conferred enhanced resistance to F. oxysporum. These mutants also have enhanced resistance to a leaf fungal pathogen (Alternaria brassicicola) and an aphid pest (Myzus persicae), but not to the bacterial leaf pathogen Pseudomonas syringae. The causal alleles in these mutants were found to have defects in the ESR1 interacting protein partner RNA Polymerase II Carboxyl Terminal Domain (CTD) Phosphatase-Like1 (CPL1) and subsequently given the allele symbols cpl1-7 and cpl1-8. These results define a new role for CPL1 as a pathogen and pest susceptibility gene. Global transcriptome analysis and oxidative stress assays showed these cpl1 mutants have increased tolerance to oxidative stress. In particular, components of biotic stress responsive pathways were enriched in cpl1 over wild-type up-regulated gene expression datasets including genes related to defence, heat shock proteins and oxidative stress/redox state processes

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

Proposition of a SNP set to replace SSRs for standardized cultivar identification in apple

Apple is one of the most important fruit crops grown in temperate regions. Ex situ conservation of apple genetic resources mainly relies on grafted trees maintained in orchards, which can be space- and labor-intensive. To help collection managers to compare their germplasm with others’ at both the national and international levels, Malus UNiQue genotype codes (MUNQ) were proposed for all accessions sharing the same genetic marker profile. These genetic profiles were initially obtained using a standardized set of 16 SSRs. However, an impending difficulty in SSR analysis is expected and many genetic studies nowadays use SNP markers. SNP-based MUNQ assignment is expected to be more streamlined, but just like for SSRs, it will be essential that a consistent marker set is used across studies worldwide to enable comparisons among them without genotyping each new unique individual with a more expensive array. For this reason, we developed a set of 96 SNP markers that can unequivocally distinguish cultivars in apple collections and detect redundancy faster at low cost. To support this approach, we started from genome-wide SNP genotypic data obtained with the apple 20K and/or 480K arrays for 2120 unique individuals, including 2036 with a MUNQ code assigned through SSR markers. A total of 182 SNPs were tested using the KASP technology in nanofluidic Integrated Fluidic Circuit (IFC) to finally choose a set of 96 SNPs ensuring that each pair of individuals among all 2120 was distinguished by at least 6 SNPs in the absence of missing data. A comparison of MUNQ code assignment for 754 newly genotyped accessions using the standardized set of 16 SSRs and the new set of 96 SNPs was performed. It was possible to group accessions with essentially unique profiles using SNP data, and the groups obtained were almost always consistent with groups obtained with SSR data. Thus, a transition from the SSR-based MUNQ system to a SNP-based MUNQ system will be possible