Function Analysis Of Necrosis-inducing Genes NPP1-like And PcF/SCR-like In Phytophthora Infestans

Potato late blight which caused by Phytophthora infestans is a general occurrenceworldwide potato disease and causes enormous economic losses. Phytophthora infestansis an oomycete. Oomycete pathogens secrete effectors to manipulate host cell structureand function and trigger host cell defenses. NPP1-like and PcF/SCR-like were two largegene families in Phytophthora infestans. Studies of NPP1-like and PcF/SCR-like havefocused on relatively few proteins. The function and pathogenic mechanism of the twofamilies remains unclear. In our study, necrosis-inducing function analysis of genePiNLP9, PiNLP30, PcF/SCR.3and PcF/SCR.13was tested.1. Candidate genes cloning and sequence analyses were performed. PiNLP9,PiNLP30, PcF/SCR.3and PcF/SCR.13were cloned, using the cDNA from P. infestansHK09-19. Full-length cDNA of PiNLP9was840bp and contained relatively conserved“GHRHDWE” heptapeptide motif. Full-length cDNA of PiNLP30was714bp with a19amino acids signal peptide and contained conserved“GHRHDWE” heptapeptide motif.PiNLP9and PiNLP30showed two conserved cysteine residues on N-terminal.Full-length cDNA of PcF/SCR.3and PcF/SCR.13were213bp and276bp respectively.Both PcF/SCR.3and PcF/SCR.13possessed a signal peptides and three predicted S-Sbridges.2. Functional analysis of the four genes were tested. PiNLP9, PiNLP30, PcF/SCR.3and PcF/SCR.13were cloned into vector pBI121, then transformed into Agrobacteriumtumefaciens and expressed in Nicotiana benthamiana by agro-infiltration method.Theresults showed that PiNLP30was able to trigger necrosis while PiNLP9did not causenecrosis. PcF/SCR.3and PcF/SCR.13were all able to trigger necrosis afer inoculated N.benthamiana leaves.3. Site-directed mutation of PiNLP30and its functional analysis was performed.The primers were designed according to the information of key residues of NLP fromFythium. Key residues were individually exchanged for alanine and expressed in N.benthamiana by agro-infiltration method. The results showed that mutatedpBI121-H122/A could cause necrosis after inoculated N. benthamiana and mutated pBI121-D122/A performed weakly activity, while pBI121-E127/A andpBI121-H122/D125/E127A mutant exhibited compromised bioactivity. These studiesindicated that the importance of amino acids at125and127loci.4. Expression pattern of PiNLP30was analyzed. The primers used in real-timefluorescent quantitative PCR were designed. Total RNA samples of infected potato tissuesamples were extracted. The first-strand cDNA was synthesized.Then real-time RT-PCRassays were carried out. The results indicated that PiNLP30was highly expressed after P.infestans zoospores inoculated into potato leaf during120hours. The first expressionpeak presented at12hours and up-regulated about260times. The second expressionpeak appeared during60-108hours and up-regulated about160-230times. The resultssuggested that PiNLP30might play a role during the whole infection stages.5. Prokaryotic expression of PiNLP9and PiNLP30was researched in our study.PiNLP9and PiNLP30was cloned into vector pET28b firstly and further transformed intoBL21(DE3). The recombinant proteins of pET28b-PiNLP9and pET28b-PiNLP30wasexpressed with the treatment of0.6mmol·L-1IPTG for6h at37℃and mainly existed ininclusion body form. The purified recombinant PiNLP30protein was obtained byNi+NTA affinity purification.