High throughput identification of genes conferring resistance or sensitivity to toxic effectors delivered by the type VI secretion system

The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner.

SUMOylation of Dorsal attenuates Toll/NF-κB signalling

In Drosophila, Toll/NF-κB signalling plays key roles in both animal development and in host defence. The activation, intensity and kinetics of Toll signalling is regulated by post-translational modifications such as phosphorylation, SUMOylation or ubiquitination that target multiple proteins in the Toll/NF-κB cascade.Here, we have generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant (SCR). Intriguingly, embryos laid by dlSCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DLSCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dlSCR animals show increase in crystal cell numbers, stronger activation of humoral defence genes, high cactus levels and cytoplasmic stabilization of DL:Cactus complexes. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (<5%) suggests that it acts to block transcriptional activation, driven primarily by DL that is not SUMO conjugated.Our findings define SUMO conjugation as an important regulator of the Toll signalling cascade, in both development and in host defense. Our results broadly indicate that SUMO attenuates DL at the level of transcriptional activation. Further, we hypothesize that SUMO conjugation of DL may be part of a Ubc9 dependant feedback circuit that restrains Toll/NF-κB signalling.

Long-term epigenetic memory of jasmonic acid-dependent immunity against herbivory

Stress exposure can have long-lasting impacts on ecologically relevant life-history traits in plants. Here, we have investigated the long-term impacts of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis thaliana. Three weeks after transient JA signalling activity in seedlings, 5-week-old plants retained induced resistance (IR) against herbivory but showed enhanced susceptibility to necrotrophic and biotrophic pathogens. Transcriptome analysis revealed that JA seedling treatment causes prolonged priming and/or up-regulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR against herbivory was dependent on MYC2/3/4 transcription factors and DNA (de)methylation pathways. Although DNA methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled defence genes, plants from JA-treated seedlings showed enrichment of differentially methylated regions at ATREP2 transposable elements (TEs). Our study points to a trans-acting mechanism whereby hypomethylated TEs mediate long-lasting epigenetic memory of JA-dependent immunity.

Deciphering Brassica plant defence responses to cabbage white butterfly egg-associated molecular patterns

Brassica plants activate a strong hypersensitive response (HR)-like necrosis underneath eggs of cabbage white butterflies, but their molecular response to eggs is poorly understood. Here, we developed a method to generate egg wash to identify potential insect egg-associated molecular patterns (EAMPs) inducing HR-like necrosis. We found that egg wash, containing compounds from Pieris eggs, induced a similar response as eggs. We show that wash of hatched eggs, of egg glue, and of accessory reproductive glands (ARG) that produce this glue, also induced HR-like necrosis, whereas removal of the glue from eggs resulted in a reduced response. Eggs of Pieris butterflies induced callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like necrosis. Finally, only washes from Pieris eggs induced defence genes and ethylene production, whereas egg wash of a generalist moth did not. Our results indicate that EAMPs are in the egg glue and that the response in B. nigra is specific to Pieris species. Our study expands knowledge on the Brassica-Pieris-egg interaction, and paves the way for identification of EAMPs in Pieris egg glue and corresponding receptor in Brassica spp.

Thinopyrum intermedium TiAP1 interacts with a chitin deacetylase from Blumeria graminis f. sp. tritici and increases the resistance to Bgt in wheat

SummaryThe biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt) is a crucial factor causing reduction of global wheat production. Wild wheat relatives, e.g. Thinopyrum intermedium, is one of the wild-used parents in wheat disease-resistant breeding. From T. intermedium line, we identified the aspartic acid protein gene, TiAP1, which involved in resistance against Bgt. TiAP1 is a secreted protein that accumulates in large amounts at the infection sites of powdery mildew and extends to the intercellular space. Yeast two-hybrid showed that it interacted with the chitin deacetylase (BgtCDA1) of Bgt. Host-induced gene silencing showed BgtCDA1 promotes the invasion of Bgt. Transcriptome analysis showed the cell wall xylan metabolism, lignin biosynthesis-related, and defence genes involved in the signal transduction were upregulated in the transgenic TiAP1 wheat induced by Bgt. The TiAP1 in wheat may inactivate BgtCDA1 deacetylation function, cause chitin oligomers expose to wheat chitin receptor, then trigger the wheat immune response to inhibit the growth and penetration of Bgt, and thereby enhance the tolerance of wheat to pathogens.

The Arabidopsis active demethylase ROS1 cis-regulates defence genes by erasing DNA methylation at promoter-regulatory regions

Active DNA demethylation has emerged as an important regulatory process of plant and mammalian immunity. However, very little is known about the mechanisms by which active demethylation controls transcriptional immune reprogramming and disease resistance. Here, we first show that the Arabidopsis active demethylase ROS1 promotes basal resistance towards Pseudomonas syringae by antagonizing RNA-directed DNA methylation (RdDM). Furthermore, we demonstrate that ROS1 facilitates the flagellin-triggered induction of the disease resistance gene RMG1 by limiting RdDM at the 3' boundary of a transposable element (TE)-derived repeat embedded in its promoter. We further identify flagellin-responsive ROS1 putative primary targets and show that at a subset of promoters, ROS1 erases methylation at discrete regions exhibiting WRKY transcription factors (TFs) binding. In particular, we demonstrate that ROS1 removes methylation at the orphan immune receptor RLP43 promoter, to ensure DNA binding of WRKY TFs. Finally, we show that ROS1-directed demethylation of RMG1 and RLP43 promoters is causal for both flagellin responsiveness of these genes and for basal resistance. Overall, these findings significantly advance our understanding of how active demethylases shape transcriptional immune reprogramming to enable antibacterial resistance.

Comparative genome analyses of Mycobacteroides immunogenum reveals two potential novel subspecies

Mycobacteroides immunogenum is an emerging opportunistic pathogen implicated in nosocomial infections. Comparative genome analyses may provide better insights into its genomic structure, functions and evolution. The present analysis showed that M. immunogenum has an open pan-genome. Approximately 36.8% of putative virulence genes were identified in the accessory regions of M. immunogenum . Phylogenetic analyses revealed two potential novel subspecies of M. immunogenum , supported by evidence from ANIb (average nucleotide identity using blast) and GGDC (Genome to Genome Distance Calculator) analyses. We identified 74 genomic islands (GIs) in Subspecies 1 and 23 GIs in Subspecies 2. All Subspecies 2-harboured GIs were not found in Subspecies 1, indicating that they might have been acquired by Subspecies 2 after their divergence. Subspecies 2 has more defence genes than Subspecies 1, suggesting that it might be more resistant to the insertion of foreign DNA and probably explaining why Subspecies 2 has fewer GIs. Positive selection analysis suggest that M. immunogenum has a lower selection pressure compared to non-pathogenic mycobacteria. Thirteen genes were positively selected and many were involved in virulence.