SUMMARY The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities.

The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before apoplastic colonization. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of 4,296 genes in P. syringae pv. syringae B728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plant Phaseolus vulgaris. Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) and in the leaf interior (apoplast), while genes involved in type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated with in planta growth were also required for maximum epiphytic or apoplastic fitness. Fourteen hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitness in planta and either the magnitude of their expression in planta or degree of induction in planta compared to in vitro conditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant–microbe interactions.

Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In Bacillus subtilis, this labile pool is sensed by equilibration with the metalloregulator Zur, as an indication of zinc sufficiency, and by CzrA, as an indication of zinc excess. Here, we demonstrate that the low molecular weight thiol bacillithiol (BSH) serves as a major buffer of the labile zinc pool. Upon shift to conditions of zinc excess, cells transiently accumulate zinc in a low molecular weight pool, and this accumulation is largely dependent on BSH. Cells lacking BSH are more sensitive to zinc stress, and they induce zinc efflux at lower external zinc concentrations. Thiol reactive agents such as diamide and cadmium induce zinc efflux by interfering with the Zn-buffering function of BSH. Our data provide new insights into intracellular zinc buffering and may have broad relevance given the presence of BSH in pathogens and the proposed role of zinc sequestration in innate immunity.