Roles of microbiota in autoimmunity in Arabidopsis.

Over the past three decades, researchers have isolated plant mutants that display constitutively activated defense responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. From a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes a S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, displaying lesions, and having a high basal level of defense gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several "lesion mimic"-type autoimmune mutants in Arabidopsis. Interestingly, autoimmune phenotypes caused by mutations in NLR genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of two classes of autoimmunity (microbiota-dependent vs. microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom.

Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum.

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1.

Many Gram-negative bacteria use a type III secretion system (T3SS) to establish associations with their hosts. The T3SS is a conduit for direct injection of type-III effector proteins into host cells, where they manipulate the host for the benefit of the infecting bacterium. For plant-associated pathogens, the variations in number and amino acid sequences of type-III effectors, as well as their functional redundancy, make studying type-III effectors challenging. To mitigate this challenge, we developed a stable delivery system for individual or defined sets of type-III effectors into plant cells. We used recombineering and Tn5-mediated transposition to clone and stably integrate, respectively, the complete hrp/hrc region from Pseudomonas syringae pv. syringae 61 into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. We describe our development of Effector-to-Host Analyzer (EtHAn), and demonstrate its utility for studying effectors for their in planta functions.