Date Published: January 2, 2014
Publisher: Public Library of Science
Author(s): Tatiana S. Mucyn, Scott Yourstone, Abigail L. Lind, Surojit Biswas, Marc T. Nishimura, David A. Baltrus, Jason S. Cumbie, Jeff H. Chang, Corbin D. Jones, Jeffery L. Dangl, Sarah R. Grant, Frederick M. Ausubel.
Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better insight into the evolution of virulence across an evolutionarily diverse pathogen species.
Many Gram-negative bacteria attach to host cells and translocate effector proteins into them via type III secretion systems (T3SS). Such systems are necessary for pathogenesis, are horizontally transferred across species, and are accompanied by dynamically evolving repertoires of type III effector (T3Es) genes , . The T3SS is essential for Pseudomonas syringae pathogens to thrive in plant tissues. P. syringae represents an excellent example of the plasticity of T3E repertoires . Despite a collectively broad host range for the species, individual isolates of P. syringae typically display pathogenic potential on a limited set of plants and either elicit immune responses, or simply fail to thrive on other plant species. Strains can be isolated from diseased plants, as epiphytes from healthy plants , and from various environmental sources .
P. syringae is a broadly distributed and agronomically important pathogen of many plant species. Full virulence for many strains within this species requires expression of genes induced by the sigma factor HrpL, but the HrpL regulon has only been systematically surveyed using microarrays in PtoDC3000,  and to a limited extent by promoter probe studies in a few strains , . Using RNA-seq, we successfully defined HrpL regulons across six phylogenetically diverse strains. We benchmarked our data set with previous transcriptional studies of two reference genomes – (Table 2) and with qRT-PCR analysis (Table 3, Figure S2, Figure S3). Our approach allowed us to efficiently define the HrpL regulon of multiple strains, even those for which only draft genome sequence is available. We found a plethora of non-T3E genes in these regulons and experimentally verified both newly identified T3Es and non-T3E virulence factors. Additionally, we identified a variety of mechanisms that could drive recruitment into and loss from, the main virulence regulon of P. syringae.