Molecular Mechanism Of Non-host Resistance To Phytophthora Sojae

Soybean Phytophthora root rot caused by Phytophthora sojae Kaufmann et Gerdemann, is one of the most devastating diseases in soybean production of the world. In the long run, the most economic and effective measures to control this disease is developing resistant cultivars. However, the resistance of cultivars is easily loss due to the high genetic variability of isolates. Mining durable resistance genes has become a key problem in disease resistant breeding. Unlike the gene-for-gene resistance governed by single resistance (R) genes, the non-host resistance is the most effective and lasting resistance mechanism in plant. Thus, identifying the key genes involved in the response of nonhost resistance to P. sojae may be helpful to using genetic engineering to create lasting and broad-spectrum resistant materials. The main purpose of this study is to find genes involved in signal recognition and resistance response of non-host Arabidopsis and Nicotiana to P. sojae, and give new evidence to explain the molecular mechanism of non-host resistance to P. sojae. The main results were as follows:1. High virulence variability occurred during P. sojae asexual reproduction, the value of virulence variability was determined by both P. sojae isolates and host genotype. The response of Arabidopsis and Nicotiana leaf on P. sojae infection showed they have different resistance mechanism. Unlike no visible symptoms were found in Arabidopsis, local necrosis was triggered in Nicotiana and incompatible soybean hosts. In vitro co-culture conditions, R sojae zoospores were pathogenic on not only non-host Arabidopsis but also compatible and incompatible host cells.2. Proteomic analysis was carried out on the systemic acquired resistance (SAR) of soybean and Arabidopsis induced by P. sojae. In soybean, protein expression patterns induced by wound, salicylic acid (SA) and P. sojae were consistent. However, although protein expression patterns in Arabidopsis induced by P. sojae and wound were consistent, there is an obvious difference between P. sojae and SA treatment. The Protein expression profiles revealed that the SAR in soybean mainly involves a SA-mediated signals, and other SA-independent defence pathway may play an important role in SAR of Arabidopsis. The functions of differentially expressed proteins in soybean and Arabidopsis induced by P. sojae are similar; most of these proteins can be classified as metabolism related enzymes and regulatory factors, suggested that the host and non-host may share the same molecular basis in SAR.3.21 Putative P. sojae elicitin gene ORF derived from EST and genome were cloned and expressed by gateway compatible Saccharomyces cerevisiae expression vector system. Functions were evaluated by inoculation Nicotiana and Arabidopsis leaf with culture supernatants from yeast transformants. The results indicated that above method is feasible to mine pathogens extracellular protein. The response of Nicotiana and Arabidopsis on ELLICITIN was different. In Nicotiana, ELICITIN trigger leaf systemic necrosis.However, only a micro-necrosis was induced in Arabidopsis and obvious thickening of the cell wall were found around the inoculation sites. The discovery of most ELICITIN-like can inhibit Nicotiana leaf necrosis caused by yeast indicated that ELICITIN can suppress general elicitors-induced cell death in Nicotiana. 4. Proteins interacted with the ELICITIN and ELICITIN-like were screened from Nicotiana and Arabidopsis by yeast two-hybrid system. To ELICITIN SOJB, 14 proteins from Nicotiana and 13 proteins from Arabidopsiswere screened. To ELICITIN-like PPSE7, 11 proteins from Nicotiana and 14 proteins from Arabidopsis were screened. The results indicated that both SOJB and PPSE7 have the same binding protein in Arabidopsis or Nicotiana, suggested that SOJB and PPSE7 may share some similar biological function. In Nicotiana, ubiquitin-conjugating protein and peptidase were found binding with SOJB but not PPSE7. The functions of them were involved in protein degradation, which accord with the effect of SOJB on Nicotiana in this experiment. Both SOJB and PPSE7 can bind the transmembrane transport protein, SOJB with copper transporter protein, and PPSE7 with water channel protein. The results suggested that the target of SOJB and PPSE7 may be located in cytoplast.Two kinases interacted with SOJB and PPSE7 were selected from Arabidopsis. PPSE7 with ATP-binding kinase and SOJB with putative receptor kinase.To my knowledge, this is the first time a receptor-binding kinase interacted with ELICITIN was found in Arabidopsis.5. For further functional analysis on the candidate genes, gateway compatible RNAi expression vector containing 21 target genes were constructed.