Cloning and Function Analysis of Necrosis- inducing Gene PcF/SCR.1in Phytophthora infestans

Phytophthora cactorum-fragaria(PcF)proteins, distributing in Phytophthora only, are considered to be important virulence factors. There are 20 PcF/SCR(small cysteine- rich)- like genes which have been found by genomics research in Phytophthora infestans, and no necrosis- inducing function confirmation forplants yet. In this study, one of them was selected to testify its necrosis- inducing function, which was predicted to have 4 disulfide bonds in the mature protein, hereby named PcF/SCR.1(GenBank: XM_002902885). The full-length cDNA of PcF/SCR.1 was obtained by RT-PCR with the length of 336 nt and then inserted into binary expression vector pBI121 to produce recombinant plasmid pBI121- PcF/SCR.1. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 and analyzed necrosisinducing function by heterologous expression in Nicotiana benthamiana. The results showed that PcF/SCR.1caused leaf yellow at 5 days post induction(dpi) and shrinkage and necrotic symptoms at 7 dpi, indicating that the gene was able to induce plant necrosis. Furthermore, sequence alignments among PcF/SCR.1 and PcF protein showed that there were 3 disulfide bonds(between C89 and C99, C68 and C100, C73 and C104),similar to that of PcF proteins, and 1 additional potential disulfide bonds(between C88 and C110) existed only in PcF/SCR.1. All those disulfide bonds were predicted to important for maintaining protein structures and function. In order to furtherly understand the necrosis- inducing function of PcF/SCR.1, site directed mutagenesis that replaced 4 cysteines with alanine at amino acid sites at 99, 100, 104 and 110 of PcF/SCR.1was used to destroy each disulfide bridge. The resulted plasmids then heterologously expressed in N.benthamiana. The agro-infiltration assay results showed that a cysteine point mutation at aa 110, or double mutations at aa 99 and aa 110 had no effects on the necrosis-inducing function of PcF/SCR.1. However, 3cysteine mutations(C99/100/110) or 4(C99/100/104/110) of PcF/SCR.1 only caused slightly yellow symptoms in leaves in contrast to that of PcF/SCR.1, suggesting that the necrosis- inducing function was compromised. For better understanding of the gene expression patterns during P. infestans infection potato(Solanum tuberosum), Real-time RT-PCR was applied to analyze gene expression levels at different infection stages. The results showed that gene PcF/SCR.1 was up- regulated after P. infestans inoculation in potato leaves and at 48 h highly expressed about 80 times compared with control. The expression pattern indicated that gene PcF/SCR.1 played a role in an infection process, probably through suppressing host defenses and facilitating colonization of the pathogen. The study provides useful data to understand the role of the gene during P. infestans pathogenesis and valuable references for researching gene functions of other members in PcF/SCR-like gene family.