Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions

Authors: LAM, HANH - Cornell University; CHAKRAVARTHY, SUMA - Cornell University; WEI, HAI-LEI - Cornell University; BUINGUYEN, HOANGCHUONG - Cornell University; Stodghill, Paul; Swingle, Bryan; COLLMER, ALAN - Cornell University; Cartinhour, Samuel

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/28/2014
Publication Date: 8/29/2014
Citation: Lam, H.N., Chakravarthy, S., Wei, H., Buinguyen, H., Stodghill, P., Swingle, B.M., Collmer, A., Cartinhour, S.W. 2014. Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions. PLoS Pathogens. 9(8):e106115. DOI: 10.1371/journal.pone.0106115.

Interpretive Summary: The protein, HrpL, is necessary for the bacteria, Pseudomonas syringae, to cause bacterial speck in tomatoes, halo blight in beans, and various bacterial diseases in soybeans, rice, millet and many other agriculturally significant crops. HrpL has been studied extensively, and it is known that HrpL regulates genes involved in the construction of the type III secretion system (T3SS) and the deployment of disease-causing agents directly into plant cells. However, it is not known whether all the genes that HrpL regulates have been identified. To search for novel HrpL-regulated genes, we used advanced DNA and RNA sequencing technology to identify genes that are accompanied by HrpL DNA binding sites. The analysis recovered 20 new potential HrpL DNA binding sites, which regulate genes with many different functions. One gene produces a protein that is injected into plant cells during the infection process. It is not yet known what roles the other new genes play in plant disease. Many of the new genes can be found in other bacteria, including those that do not cause plant disease. With the identification of the 20 new promoters, the list of genes regulated by HrpL is probably approaching saturation. These results will help biologists to better understand how bacteria cause disease in plants and help plant breeders to develop better disease resistant crops.

Technical Abstract: The type III secretion system (T3SS) is required for virulence in the gram-negative plant pathogen Pseudomonas syringae pv. tomato DC3000. The alternative sigma factor HrpL directly regulates expression of T3SS genes via a consensus promoter sequence, often designated as the “hrp promoter.” Although the HrpL regulon has been extensively investigated in DC3000, it is not known whether additional regulon members remain to be found. To systematically search for HrpL-regulated genes, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and bulk mRNA sequencing (RNA-Seq) to identify HrpL-binding sites and likely hrp promoters. The analysis recovered 73 sites of interest, including 20 sites that represent new hrp promoters. The new promoters lie upstream of a diverse set of genes encoding potential regulators, enzymes and hypothetical proteins. In our data, PSPTO_5633, a conserved protein of unknown function, is the only new HrpL regulon member that is potentially an effector. Deletion of this gene in a D28E background, from which most other effectors have been removed, has a modest effect on in planta growth. Deletions in several other new regulon members, including PSPTO_5633, PSPTO_0371, PSPTO_2130, PSPTO_2691, PSPTO_2696, PSPTO_3331, and PSPTO_5240, in either DC3000 or 'hopQ1-1 backgrounds, do not affect the hypersensitive response or in planta growth of the resulting strains. Many new HrpL regulon members appear to be unrelated to the T3SS, and orthologs for some of these can be identified in numerous non-pathogenic bacteria. With the identification of 20 new hrp promoters, the list of HrpL regulon members is approaching saturation and most likely includes all DC3000 effectors.