Functional Dissection Of The StPOTHR1 Gene In Potato Against Phytophthora Infestans

Potato(Solanum tuberosum L.)is currently the fourth most important global staple crop after wheat,rice,and maize.Late blight,which is caused by the oomycete pathogen Phytophthora infestans,is the most devastating diseases of potato.To survive infection,potato plants have evolved a two-branched innate immune system to detect and defend against P.infestans,including pathogen associated molecular patterns(PAMPs)triggered immunity(PTI)and effector-triggered immunity(ETI).To minimize infection pressure,plants can activate the expression of defense-related genes,occasionally resulting in a typical disease resistance response known as a rapid hypersensitive response(HR)that can effectively restrain the expansion of the pathogen.Our previous study identified and cloned a HR associated gene which termed as StPOTHR1,StPOTHR1 can be activated by P.infestans,mechanical injury,salt stress,and jasmonic acid.However,the mechanism underlying how StPOTHRl functions in potato late blight resistance remains largely unknown,and it is not yet clear if this gene is involved in HR.In present study,the relation between StPOTHR1 and HR,the functional mechanism of StPOTHR1 in potato resistance against P.infestans,and the immunity signaling pathways mediated by StPOTHR1 were investigated,the results provide new thoughts and potential gene resources for the rational design of potato breeding for durable resistance against P.infestans.The major results were as follows:1.W8-G20,a transgenic P.infestans variant of that retains similar pathogenicity to the wild-type W8 but that constitutively expresses GFP as a marker of visualization,was provided a technological means with intuition and convenience to conduct qualitative and quantitative assessments of the interactions between potato and P.infestans.After inoculation with P.infestans W8-G20,a significantly slower disease development was observed in the race-nonspecific resistant potato clone 386209.10 than in the susceptible variety E-potatol(E1),the fluorescence intensity on the leaves from El was significantly higher than that in 386209.10 after infection,as comparison with El,the rapid proliferation of pathogen in 386209.10 was prominently restricted,indicating that the restraint of pathogen proliferation and colonization could be a crucial defense mechanism of horizontal resistance.2.Bioinformatics analysis characterized StPOTHRl as a NHL(NDR/HIN1-like)family gene with three conserved NHL motifs.Further phylogenetic analysis showed that StPOTHR1shares the highest identity withNtHINl andAtNHL3 genes,suggesting that StPOTHRl may be involved to biological process such as plant HR and/or plant immunity.By means of in situ hybridization and transcripts quantification,it was found that the StPOTHRl gene was specifically expressed in inoculation sites,where HR occurs in potato resistant variety E-potao3(E3)as challenged by P.infestans.Consistent with a role for StPOTHRl in HR in response to P.infestans,the StPOTHRl expression was not detected in locations on the same leaflet apart from the HR lesion,implying that the expression of StPOTHR1 was tightly related to HR,and StPOTHRl may play crucial role in potato immunity.3.The expression pattern analysis of StPOTHRl during the different interactions among potato and P.infestans,such as compatible interaction(susceptibility),incompatible interaction(resistance),and horizontal resistance(non-race-specific resistance),indicated that the abundance of StPOTHRl transcripts was strongly associated with the resistance levels of particular potato varieties against P.infestans.A rapid response of StPOTHRl and maintaining higher levels of StPOTHRl transcription were observed in potato resistant variety E3,a low level of expression but weak upregulation at late stage of infection in the late blight susceptible variety El,and an intermediate level of expression in potato clone 386209.10 that harbors race-nonspecific resistance to late blight.These findings indicated that the abundance of StPOTHRl transcripts were positively correlated with intensity of potato late blight resistance.4.Over expression of StPOTHRl in the susceptible potato variety E1 significantly reduced the amount of pathogen sporangia and enhanced resistance to P.infestans in a type of response which resembled that of the non-specific resistant clone 386209.10.However,silencing StPOTHRl in race-specific resistant potato E3 showed no obvious impact on resistance.Further transient silencing the ortholog of StPOTHRlin Nicotiana benthamiana(NbPOTHRl)by VIGS did not compromise the HR triggered by co-expression of R3a/Avr3a,Stollpio,Vnt-1/Avrvnt-1,Pto/AvrPto,Cf4/AvrCf4,or Cf9/AvrCf9.These results therefore suggest that the contribution of StPOTHRl to potato late blight resistance could be independent of known R gene mediated pathways.5.The expression pattern of POTHR1 during the HR triggered by co-expression of R3alAvr3a,Sto/Ipio,Vnt-1/Avrvnt-1,Pto/AvrPto,Cf4/AvrCf4,or Cf9/AvrCf9 found that a specific response merely occurred in HR triggered by co-expression of Pto/AvrPto.Moreover,the expression of POTHR1 was induced by the P.infestans effector INF1 and bacterial PAMPs flg22 which can trigger PTI,the POTHR1 could regulate the expression of the early-induced genes,indicated POTHR1 may involve in the crosstalk between PTI and ETI.6.The coding sequences of StPOTHRl were cloned in-frame with a N-terminal GFP fusion and transiently expressed in N.benthamiana.GFP-StPOTHR1 protein was proved to locate in the plasma membrane.Immunoblots revealed that the GFP fusion protein was intact,indicating that the fluorescence accurately reflects the localization of StPOTHRl.However,the immunoreactive band of GFP-StPOTHRl was larger than the predicted molecular mass.We reasoned that the increased size of the fusion protein was potentially due to the presence of post-translational modifications(PTMs)in StPOTHR1.By performing LC-MS/MS,it was found that StPOTHR1 has multiple PTMs,and these PTMs can likely account for the larger observed size of GFP-StPOTHR1.To the best of our knowledge,this study represents the first successful effort to systematically identify the PMTs of a NHL family protein.The further research on the functions of these PTMs sites will improve our knowledge of the plant immunity.7.IP-LC-MS/MS analysis showed that NbMKK5L,a component of the MAP kinase signaling cascade,is a potential interaction partner of StPOTHRl.The interaction between NbMKK5L and StPOTHR1 was further confirmed by co-IP assay,a finding implying that the enhancement of resistance against P.infestans that resulted from the overexpression of StPOTHR1 is related to MAP kinase signaling.We have also successfully developed an Escherichia coli expression system to easily generate large amounts of purified StPOTHR1 protein,a requirement to further investigate the regulatory mechanism between StPOTHR1 and NbMKK5L in vitro;for instance,to determine whether the kinase NbMKK5L can directly phosphorylate StPOTHR1 and the like.