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, which 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 NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host’s pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.

Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because these subcellular structures lack a delineating membrane, determining their compositions is difficult. Here we describe a proximity-biotinylation approach for capturing the RNAs of the condensates known as processing bodies (PBs) in Arabidopsis (Arabidopsis thaliana). By combining this approach with RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal conditions and heat stress. PBs showed a much more dynamic RNA composition than the total transcriptome. RNAs involved in cell wall development and regeneration, plant hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA-binding proteins and the liquidity of PBs modulated RNA recruitment, while RNAs were frequently recruited together with their encoded proteins. In PBs, RNAs follow distinct fates: in small liquid-like PBs, RNAs get degraded while in more solid-like larger ones, they are stored. PB properties can be regulated by the actin-polymerizing SCAR (suppressor of the cyclic AMP)-WAVE (WASP family verprolin homologous) complex. SCAR/WAVE modulates the shuttling of RNAs between PBs and the translational machinery, thereby adjusting ethylene signaling. In summary, we provide an approach to identify RNAs in condensates that allowed us to reveal a mechanism for regulating RNA fate.

SUMMARY: Exo70B1 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.

Fluorescent protein-tagged bacterial strains are widely used tools for studying host-pathogen interactions and microbial dynamics. In this study, we developed and characterized Vibrio harveyi strains genetically modified to express green fluorescent protein (GFP) and red fluorescent protein (RFP). These strains were constructed using triparental mating and evaluated for phenotypic, genomic, and virulence attributes. Genomic analyses revealed strain-specific variations, including mutations in key regulatory and metabolic genes, such as luxO and transketolase. While plasmid acquisition imposed metabolic costs, resulting in altered growth and antibiotic sensitivities in certain transconjugants, others demonstrated robust phenotypic stability. Virulence assays using gilthead seabream larvae revealed that most tagged strains retained moderate pathogenicity, with visualization of co-infections highlighting the potential for studying strain-specific interactions. Furthermore, fluorescent microscopy confirmed the successful colonization and localization of tagged bacteria within host tissues. These findings underscore the utility of GFP- and RFP-tagged Vibrio harveyi as versatile tools for infection dynamics, offering a foundation for future research on strain interactions and pathogen-host relationships.

CircRNAs are widely present in plants, yet there have been no reports on the role of circRNA in the regulatory mechanisms of interactions between rice and pathogenic bacteria. As the sequence of circRNAs matches the parent gene except for the back splicing site, there is no feasible way to specifically knockdown circRNAs in plants. We identified a circRNA, named Os-circANK, derived from an ankyrin repeat protein, which was significantly downregulated post Xanthomonas oryzae pv. oryzae ( Xoo ) inoculation. We have created a CRISPR-Cas13d system to specifically knockdown a plant circular RNA, resulting in genetically edited rice with a targeted decrease in Os-circANK expression. Os-circANK was found to function as a sponge for Osa-miR398b, suppressing the cleavage of OsCSD1/OsCSD2 by Osa-miR398b, leading to a reduction in ROS levels following Xoo infection and a negative regulation of resistance to bacterial blight in rice. Our findings demonstrate that Os-circANK inhibits the resistance to bacterial blight in rice via the Osa-miR398b/OsCSD1/OsCSD2 pathways.

Cellular condensates are usually ribonucleoprotein assemblies with liquid– or solid–like properties. Because they lack a delineating membrane, the compositional determination of condensates is laborious. Here we set up a pipeline for proximity–biotinylation–dependent capture of RNA to investigate the RNA composition of the condensate in Arabidopsis known as the processing bodies (PBs). Using this pipeline together with in situ protein–protein interaction and RNA detection, in silico , and high–resolution imaging approaches, we studied PBs under normal and heat stress conditions. The composition of PBs in RNAs is much more dynamic than that of the total transcriptome. RNAs involved in cell wall development and regeneration, hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA binding proteins and liquid–to–solid phase transitions modulated specificity of RNA recruitment in PBs. Surprisingly, RNAs were sometimes recruited together with their encoded proteins. In PBs RNAs follow distinct fates, with small liquid-like PBs modulating RNA decay while larger ones storage. The size and properties of PBs are regulated by the actin polymerization cAMP receptor (SCAR)–WASP family verprolin homologous (WAVE) complex. SCAR/WAVE modulates signaling by shuttling RNAs between PBs and the translational machinery adjusting the ethylene signaling pathway. Heat stress leads to the storage of immunity–related RNAs in PBs by reducing PBs dynamics, suggesting why processes such as immunity malfunction under heat stress. In summary, we provide a method to identify RNAs in condensates which allowed us to reveal a mechanism for RNA fate regulation.