| On a global scale, potato(Solarium tuberosum L.) is the fourth important food crop which plays a vital role in both industry and agriculture. Late blight, the most destructive disease of potato, is caused by Phytophthora infestans (Mont.) de Bary which has resulted in enormous economic losses throughout the world. Two forms of genetic resistance to P. infestans in potato have been classified as qualitative and quantitative resistance. The former is associated with dominant resistance (R) genes which can be easily overcome due to rapid evolution of new virulent races of P. infestans, and the later is controlled by many interacting genes and expected to be more durable and reliable than the resistance controlled by R genes. To elucidate the genetic basis of the quantitative resistance to P. infestans, differentially expressed genes have been identified and most of them are involved in metabolism, signal transduction, transcriptional regulation and plant defense.Among these genes, a pathogenesis-related protein gene StPRp27 and a putative hypersensitive response related gene POTHR-1 have been cloned by our lab previously. Their transcripts accumulated remarkably after P. infestans attack. Mechanical wounding, osmotic stress and jasmonic acid could also induce their expression. Therefore, it is suggested that StPRp27 and POTHR-1 may be involved in potato resistance to late blight. However, their functions in the disease resistance remains to be elucidated. To look into this aspect, transient expression and silencing as well as the genetic thansformation of the genes were conducted and the main findings are as follows:1. To determine whether StPRp27 confers resistance to P. infestans, wild type plants of N. benthamiana, which are susceptible to P. infestans, were used to transient express StPRp27, POTHR-1, Rpi-R3a (positive control for P. infestans isolate 99189), Rpi-sto (positive control for P. infestans isolate PY23) and abpt (a non-functional R gene which was used as negative control), respectively. The results showed that expression of StPRp27 and POTHR-1 enhanced the resistance to P. infestans, preliminarily indicate the involvment of these two genes in plant late blight resistance.2. To test if the R gene mediated resistance or HR requires StPRp27 or POTHR-1 gene, the two genes were silenced in potato and transgenic N. benthamiana plants respectively using the virus induced gene silencing system (VIGS). The results showed no effects of the suppression of StPRp27 or POTHR-1 gene on either the R gene (Rpi-blbl and Rpi-blb2) mediated defense or the recognition between the R gene and its corresponding effector, demostrating that the resistance governed by StPRp27 and POTHR-1 is independent of the pathway mediated by the known R genes.3. To further verify whether or not StPRp27 could contribute to potato resistance to P. infestans, StPRp27 gene was over-expressed and RNAi suppressed in potato cv. E-potato 3 (E3) by Agrobacterium-mediated transformation. RT-PCR analysis of the transgenic plants showed that the expression of StPRp27 in over-expression transgenic lines are much higher than that in wild type E3, whereas significant silencing was observed in the RNA interference transgenic lines. Selected transgenic plants were subjecyed to late blight resistance assessment and lower LGRs (lesion growth rate) were obtained from over-expression transgenic plants compared with the control. However, there were no significant difference in LGR between StPRp27 silenced lines and control plants. The results suggested that StPRp27 is involved in potato defense against P. infestans and a post-transcriptional regulation may be critical for its function.4. The StPRp27 gene was inserted into the vector pGEX-6P-1 and transferred into E. coli BL21(DE3). The fusion protein StPRp27-GST was purified and used in the inhibition experiment. Results showed that the fusion protein could inhibit the germination of spores of P. infestans implying an antifungal activity of StPRp27 and the gene may be in the downstream of the resistance pathway.. To analyze the localization of the StPRp27 protein in plant cells, the GFP and StPRp27 were fused and transiently expressed in onion epidermal cells. The result showed that StPRp27-GFP localized in the intracellular space. It is reasonable to suggest that StPRp27 protein has function of inhibiting the growth of P. infestans in plant cells. The diploid potato parents SH83-92-488 (SH) and RH89-039-16 (RH) and their 84 F1 pedigree clones were used to map StPRp27 and POTHR-1 in potato genome. The CAPS markers specificly designed for the StPRp27 and POTHR-1 were mapped to the existing UHD database and located on chromosomes I and chromosomes X, respectively. |
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