Functional analysis for this locus Functional Analysis of PR10 genes in soybean Maria Laura Malvino1, David J. Neece2, Steven J. Clough1,2 1 University of Illinois at Urbana-Champaign;2 USDA/ARS, Urbana IL Summary Figure 2 This research is designed to characterize soybean genes that respond to pathogens like Sclerotinia sclerotiorum and Pseudomonas syringae. One such class of genes are PR10 genes. A recent publication (Choi D. S., Hwang I. S., Hwang B. K. 2012. Plant Cell 24: 1675-1690) shed some light on this topic by showing that PR10s may interact with leucine-rich repeat protein 1 (LRR1) to form a complex that is responsible for cell death-mediated defense signaling, showing that some pathogenesis-related proteins might be involved in signaling. We have selected 6 candidate genes of the PR10 family based on in-house microarray studies (in-house database) and blast searches. We are obtaining full-length cDNA of the selected pathogen-responsive PR10 genes and are cloning them into Agrobacterium overexpression vectors for Arabidopsis and soybean transformation. RNAi constructs are also being made for transformation into soybean. The transgenic plants will be assayed for altered responses to pathogens. Additionally, we are studying the response of PR10 promoters by studying GFP expression off the constitutive promoters Gmubi and PR10 promoters, both provided by the Finer lab at The Ohio State University. This ongoing research project will increase our understanding of promoter function, allow us to detail specificity of gene expression and verify effectiveness of PR10 in defense responses to pathogen infection or pests, lead to the development of markers that are associated with resistance, and allow us to generate a collection of soybean promoters for basic research and possible transgenics with enhanced resistance. Clone RT-PCR Product into pBin-T vector Grow colonies in kanamycin resistance plates RT-PCR Check orientation by PCR with a primer from the gene of interest and a M13 primer. Afterwards, send to sequence Sequentially plant seeds in kanamycin plates until the 3rd generation in order to get homozygous plants. Conduct disease studies. Grow colony with right orientation in liquid media overnight and then extract plasmid with a purification kit Transplant kanamycin resistant plants into soil Electroporate ligation mix into Agrobacterium tumefacians Seeds Selection on Kanamycin plates Floral Dip transformation Check full CDS sequence with PCR-gel Culture Agrobacterium Note: pBin-T is a modified version of the pBin-mgfp5-ER vector, and contains an AhdI cloning cassette which allows for direct cloning of PCR products. III. Evaluation of PR10 expression: promoters Transformation in Arabidopsis with pGmPath21 (PR10) and pGmubi promoters was done (Figure 3). pGmPath21 is a good candidate to direct pathogen-induced root-specific expression according to its differential GFP expression in soybean hairy roots and RNA-Seq data for its gene. The purpose of this evaluation is to do pathogen-induced GFP expression analysis, in order to study GFP expression driven off a soybean PR10 promoter in Arabidopsis after various biotic and abiotic challenges. Note: pCAMBIA1300 constructs pGmPath21::GmPath21 and Gmubi::GmPath21 are provided by John Finer’s Lab at The Ohio State University. Figure 3 Procedures I. Gene Selection To search for pathogen-inducible genes in soybean, we used our in-house database, Soybean Gene Expression Database (SGED), to search for genes that have the key term SAM22 (a PR10) or PR10. Afterwards, we removed some genes that were poorly matched. Then we entered the remaining GlymaID into cluster analysis selecting different experiments (symbiotic, pathogenic, chemical and others). We obtained a cluster of 6 genes, all of which appear to be responsive to pathogen infection (Figure 1). Figure 1 Gmubi-pCambia1300 construct GmPath21-pCambia1300 construct IV. Silencing of PR10 paralogs A region of homology between putative PR10 genes is being cloned and used for making an RNAi construct with the goal of silencing multiple PR10 paralogs to test the role of PR10 in soybean defense to pathogens and pests. A gateway cloning vector (from Chris Taylor, The Ohio State University) is being used for this purpose. GlymaID Functional analysis for this locus Gene Candidate 1 Glyma07g37240.2 Pathogenesis-related protein Bet v I family, response to biotic stimulus Gene Candidate 2 Glyma07g37240.3 Gene Candidate 3 Glyma17g03340.1 Gene Candidate 4 Glyma07g37270.1 Gene Candidate 5 Glyma17g03350.1 Gene Candidate 6 Glyma09g04520.1 V. Results Bacterial growth monitored in transgenic line #1 and #4 suggest an enhance resistance against both the HR and virulence strains of Pseudomonas. syringae during the first 48 hours. However, results suggest that PR-10 is not enhancing resistance to Sclerotinia sclerotiorum in Arabidopsis. Results show that soybean GmPath21 PR-10 promoter functioned in Arabidopsis in a pathogen-responsive manner, showing enhanced expression of GFP after the first hour of inoculation. Transgenic line #1 shows also phenotypic differences: larger amount of biomass (seeds and leaves) and new rosettes appearing on the fluorescent. II. Over-expression in Arabidopsis (and later soybean) Full-length cDNA of 5 pathogen-responsive genes are being cloned into an Agrobacterium overexpression vector to be transformed into Arabidopsis (and later soybean). Transgenics will be assayed for altered responses to pathogens (Figure 2). Acknowledgements This work was funded by the Fulbright Student Program, the North Central Soybean Research Program, and the USDA-ARS