For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, that is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis, and can do this without activating a plant NLR (NOD-like receptor) that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host’s pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) by blocking exocytosis of pathogenesis-related protein-1A (PR1a), callose deposition and localization of the FLS2 immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defences represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.

Background: Crop wild relatives (CWRs) contain genetic diversity, representing an invaluable resource for crop improvement. Many of their traits have the potential to help crops to adapt to changing conditions that they experience due to climate change. An impressive global effort for the conservation of various CWR will facilitate their use in crop breeding for food security. The genus Brassica is listed in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture. Brassica oleracea (or wild cabbage), a species native to southern and western Europe, has become established as an important human food crop plant because of its large reserves stored over the winter in its leaves. Brassica cretica Lam. (Bc) is a CWR in the brassica group and B. cretica subsp. nivea (Bcn) has been suggested as a separate subspecies. The species Bc has been proposed as a potential gene donor to brassica crops, including broccoli, cabbage, cauliflower, oilseed rape, etc. Results: We sequenced genomes of four Bc individuals, including two Bcn and two Bc. Demographic analysis based on our whole-genome sequence data suggests that populations of Bc are not isolated. Classification of the Bc into distinct subspecies is not supported by the data. Using only the non-coding part of the data (thus, the parts of the genome that has evolved nearly neutrally), we find the gene flow between different Bc population is recent and its genomic diversity is high. Conclusions: Despite predictions on the disruptive effect of gene flow in adaptation, when selection is not strong enough to prevent the loss of locally adapted alleles, studies show that gene flow can promote adaptation, that local adaptations can be maintained despite high gene flow, and that genetic architecture plays a fundamental role in the origin and maintenance of local adaptation with gene flow. Thus, in the genomic era it is important to link the selected demographic models with the underlying processes of genomic variation because, if this variation is largely selectively neutral, we cannot assume that a diverse population of crop wild relatives will necessarily exhibit the wideranging adaptive diversity required for further crop improvement.

Nucleotide-binding and leucine-rich repeat receptors (NLRs) are intracellular plant immune receptors that recognize pathogen effectors secreted into the plant cell. Canonical NLRs typically contain three conserved domains including a central nucleotide binding (NB-ARC) domain, C-terminal leucine-rich repeats (LRRs) and an N-terminal domain. A subfamily of plant NLRs contain additional noncanonical domain(s) that have potentially evolved from the integration of the effector targets in the canonical NLR structure. These NLRs with extra domains are thus referred to as NLRs with integrated domains (NLR-IDs). Here, we first summarize our current understanding of NLR-ID activation upon effector binding, focusing on the NLR pairs Pik-1/Pik-2, RGA4/RGA5, and RRS1/RPS4. We speculate on their potential oligomerization into resistosomes as it was recently shown for certain canonical plant NLRs. Furthermore, we discuss how our growing understanding of the mode of action of NLR-ID continuously informs engineering approaches to design new resistance specificities in the context of rapidly evolving pathogens.

Ralstonia solanacearum depends on numerous virulence factors, also known as effectors, to promote disease in a wide range of economically important host-plants. Although some of these effectors have been characterized, none has been shown to target the host secretion machinery so far. Here, a screening was performed, using an extended library of NLR plant immune receptors integrated domains (IDs), to identify new effector targets. The results uncovered that the core effector RipE1, of the R. solanacearum species complex, among other targets, associates with Arabidopsis exocyst component Exo70B1. RipE1, in accordance with its predicted cysteine protease activity, cleaves Exo70B1 in vitro between its sequence and is able to promote Exo70B1 degradation in planta. RipE1 enzymatic activity additionally results in the activation of TN2-dependent cell death. TN2 is an atypical NLR that has been proposed to guard Exo70B1. Despite the fact that RipE1 has been previously reported to activate defense responses in model plant species, we present here a Nicotiana species, in which RipE1 expression does not activate cell death. Overall, this study uncovers a new RipE1 host target, while providing evidence and novel tools to advance in-depth studies of RipE1 and homologues effectors.

We present here the draft genome sequences of type/pathotype strains for three Xanthomonas species and pathovars with different host specificities, the Hedera helix L. pathogen Xanthomonas hortorum pv.

SUMMARY Plant immunity largely relies on intracellular nucleotide‐binding domain leucine‐rich repeat (NLR) immune receptors. Some plant NLRs carry integrated domains (IDs) that mimic authentic pathogen effector targets. We report here the identification of a genetically linked NLR‐ID/NLR pair: BnRPR1 and BnRPR2 in Brassica napus. The NLR‐ID carries two ID fusions and the mode of action of the pair conforms to the proposed “integrated sensor/decoy” model. The two NLRs interact and the heterocomplex localizes in the plant‐cell nucleus and nucleolus. However, the BnRPRs pair does not operate through a negative regulation as it was previously reported for other NLR‐IDs. Cell death is induced only upon co‐expression of the two proteins and is dependent on the helper genes, EDS1 and NRG1. The nuclear localization of both proteins seems to be essential for cell death activation, while the IDs of BnRPR1 are dispensable for this purpose. In summary, we describe a new pair of NLR‐IDs with interesting features in relation to its regulation and the cell death activation.

The exocyst complex subunit protein Exo70B1 plays a crucial role in a variety of cell mechanisms including immune responses against pathogens. The calcium dependent kinase 5 (CPK5) of Arapidopsis thaliana, phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestis effector XopP, compromises Exo70B1 and bypasses the host's hypersensitive response (HR), in a way that is still unclear. Herein we designed an experimental approach based on biophysical, biochemical and molecular assays, based on structural and functional predictions, as well as, utilizing Aplhafold and DALI online servers respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP is very stable in high temperatures, while the AtExo70B1 appeared to be phosphorylated at XccXopP expressing transgenic Arabidopsis. XccXopP reveals similarities with known mammalian kinases, and phosphorylates AtExo70B1 at Ser107, Ser111, Ser248, Thr309 and Thr364. Furthermore, XccXopP protects AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that, XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host's protein target AtExo70B1, but also protects the latter from the innate AtCPK5 phosphorylation, to bypass the host's immune responses.

SUMMARYExo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium‐dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP‐expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405.