Identification Of The Function Of Four Genes Encoding The RxLR Secreted Protein In Phytophthora Infestans

Potato late blight causing by Phytophthora infestans(Mont.) de Bary is considered as the first international crop diseases that caused a serious threat by resulting in dramatically reduced the yield of potato. During the stage of co-evolution between pathogens and plant, P.infestans secreted numerous RxLR effectors which acted as weapons to plays different pathogenic roles to successfully infect the potato. Oppositely, as the "shield" components of plant immune system, the current cloned resistance genes for late blight were identified to recognize its cognate avirulence genes that were belonged to Rx LR secreted proteins. To be an autotetraploid crops and self-incompatibility, only very limited resistance resources of wild species could introduce into cultivars by traditional hybridization in potato. Therefore, it becomes one of the important prevention strategies that identify late blight resistance genes from wild germplasm resources and make transgenic potato for late blight resistance with engineering techniques.Solanum nigrum is a non-host solanum plant with very good resistance performance to P. infestans. It is predicted to habour many resistance genes of late blight resistance. In this study, a RXLR effector pools was constructed with the HLJ1, a strain of A1 anastomosis group isolated from Heilongjiang province of China.Then the screening and the functional analysis of candidate avr genes were performed on collection of S.nigrum. The general results will help to clone the R genes and to exclusive the fundamental of non-host resistance. Currently, the research achievement following:1. With the reference draft genome sequences of P. infestans isolate T30-4, we cloned and constructed the total number of 251 genes of RXLR effector into pGR106, a transient expression vector, to generate a pools for P. infestans isolate HLJ1. We examined the sequence polymorphisms of all cloned RXLR effectors between P.infestans isolates HLJ1 and isolates T30-4. Most of them are equal identity on amino acid sequence, while some of them are different among the sequence with one to several amino acid substitutions. Only eight of them show no amplification among on other detective strains.2. To investigate the function of RxLR effectors, we screened the effector on both Nicotiana benthamiana and S. nigrum with PVX-mediated transient expression system. Three out of 125 cloned Rx LR genes were identified to cause hypersensitive response(HR) on S. nigrum SN0022, while not caused HR on N. benthamiana. They are predicted to the candidate avr genes for resistance genes carried in SN0022, included with Pi15718, Pi15556 and Pi21190. By fluorescence quantitative RT-PCR analysis of three candidate avr genes, they were shown significantly changes during the infection process. Through analiysis three Rx LR effector molecule truncated functional domains, all of them are shown that C- terminal played important role in inducing HR on Solanum nigrum.3. To further investigate the allele sequence of Pi15178, the results showed that they are divided into two subtypes from total ten strains of P. infestans, represented as Pi15178HLJ1 and Pi1517888069. All members of Pi15178HLJ1 could elicit HR on SN0022 and other collections of S. nigrum, but not on SN005. And the member of Pi1517888069 failed to elicit the HR on S. nigrum. This is indicated that the amino acid changes influenced the elicitor functions. More interestingly, both Pi15178HLJ1 and Pi1517888069 share the identical sequence on C-terminal which is enough to elicit the HR on SN0022, suggesting that the N-terminal of Pi1517888069 carries the repressor domain to suppress the elicitor functions. With a series of truncated assay from N-terminal, the repressor domain had been narrowed down in the range of 45 to 52 amino acid of Pi1517888069. At the same time, stable transgenic plant and yeast two hybridization were generated to further evaluate the function of the candidate avr.4. For the candidate avr gene Pi15556, it was identified to highly conserve to share the identical sequence among 15 strains of P. infestans. Also it could elicit the HR on most of collections of S.nigrum,except for SN004, SN005 and SN008. To investigate the function in P. infestans, two mutation strains were generated by RNAi technique, which show 61.9%(M-1) and 34.7%(M-2) relative expression levels compared with wild type, respectively. Biological analysis of mutants suggested that Pi15556 does not affect the colony morphology, mycelial growth and sporangium morphology. It seems to influence the production of sporangium and pathogenicity which need to further study.5. The similarinduced the HR were observed with Pi21190. It was predicted to avr gene which could only induce HR on some collections of S.nigrum, such as SN0022. Althoug the allele of Pi21190 shared difference of amino acid polymorphisms. They could induce the HR on SN0022 for all tested strains. This was suggested that Pi21190 might be conserved Rx LR effectors during the evolution.6. At the same time, a Rx LR effector named Pi14684 was screened out for suppressing the HR caused by INF1, a well-known elicitor for PTI in oomycetes. Sequence mining described several homologs of Pi14684 in difference strains of P. infestans, which shares 1 to 5 amino acid substitutions. After assay on NB, the E43 K was identified to involve in suppressing functions. Meanwhile, Pi14684 was only found to suppress the HR caused by INF1, but not for SFI2 and the ETI interaction between Avr3 aKI and R3 a.