Mechanism Analysis Of Potato StLTP10 In Phytophthora Infestans Resistance Regulation

Plants inevitably suffer from a variety of pathogens during their growth and development.In order to survive better,they have evolved a variety of sophisticated strategies in the long-term evolution process,including activation of mitogen-activated protein kinase(MAPK)cascade signaling pathways as well as hormone signaling pathways,outburst and scavenging of reactive oxygen species(ROS)in vivo,and expression of pathogenesis-related proteins,etc..Plant non-specific lipid transfer proteins(nsLTPs)are small basic proteins widely existing in plants.In recent years,such proteins have been shown to play important roles in plant disease resistance.As the fourth most important food crop in the world,potato(Solanum tuberosum)is often threatened by diseases.Among them,the late blight caused by Phytophthora infestans is a common devastating disease in potato production.Therefore,it is particularly important to discovery the resistance genes which can be used to cultivate new potato varieties with high yield,high quality and disease resistance.Up to now,there are only few reports about potato nsLTP and it remains unclear how nsLTP is involved in disease resistance in potato.So it is very important to study the function and mechanism of potato nsLTP in disease resistance regulation.In our research,we found that a potato nsLTP family gene,named StLTP10,was significantly up-regulated by P.infestans.And the biological function and molecular mechanism of the StLTP10 were investigated in potato disease resistance,and the main results and conclusions are as follows:(1)StLTP10 responds to the treatment of P.infestans and stress-related hormones.Analysis of induced expression pattern showed that,in addition to P.infestans,StLTP10 can also respond to multiple stress-related hormones(abscisic acid,salicylic acid,jasmonic acid),suggesting that StLTP10 may play an important role in plant stress response.(2)StLTP10 is highly conserved in evolution.StLTP10 protein contains eight conserved cysteine residues and two conserved pentapeptide domains.The first 24 amino acids at the N-terminal are signal peptide sequences.Phylogenetic analysis showed that StLTP10 was highly conserved and homologous with tomato LpLTP1.(3)StLTP10 positively regulates plant resistance to P.infestans.In order to identify the biological functions of StLTP10,StLTP10 was overexpressed in native tobacco leaves and then inoculated these leaves with P.infestans.Compared with the control,the disease resistance of StLTP10-overexpression leaves was significantly improved.Virus-induced gene silencing(VIGS)approach was used to silence StLTP10 in potato plants.Compared with TRV2::00-infiltrated potato leaves,the TRV2::StLTP10-silenced potato leaves exhibited more severe disease symptoms.StLTP10-overexpression and RNAi transgenic lines of potato plants were generated.The results of disease resistance analysis indicated that StLTP10-overexpression lines showed resistance to pathogen,while the RNAi lines showed susceptibility.Thus,StLTP10 can positively regulate plant resistance to P.infestans.(4)StLTP10 enhances the ability of ROS scavenging and expression of defense-related genes.The ROS scavenging pathway and the expression regulation of disease resistance-related genes play important roles in plant resistance to pathogens.In order to explore whether StLTP10 enhances plant resistance through these two pathways,the DAB and NBT were used first to analyze.It was found that the ROS accumulation in the leaves of StLTP10-OE plants was significantly lower than that of control,while the leaves of StLTP10-RNAi plants showed opposite phenotype.Moreover,through qRT-PCR analysis of defense-related genes,we found that StLTP10 could enhance the expression of ROS scavenging-related and defense-related genes.These results suggest that the increase of resistance to P.infestans in StLTP10-overexpressing plants may be related to the enhancements of ROS scavenging ability and expression levels of defense-related genes.(5)StLTP10 recruits ABA receptor PYL4 to plasma membrane.To explore the molecular mechanisms underlying how StLTP10 positively regulate pathogen resistance,StLTP10 was used as the bait for screening a yeast two-hybrid(Y2H)library composed of cDNA from potato leaves.Intriguingly,it was found that StLTP10 could interact with PYL4 and the interaction was then verified by using bimolecular fluorescence complementary(BiFC)experiments,luciferase(LCI)experiments,and pull-down techniques.To clarify the significance of the interaction,subcellular localization of these two proteins was first analyzed.The result showed that compared with PYL4-GFP expression alone,which was mostly localized to cytoplasm and nucleus,the PYL4-GFP protein co-expressing together with StLTP10 was mostly localized to plasma membrane.Moreover,the membrane fractionation for detection of PYL4 protein content was consistent with the above observation,indicating that StLTP10 effects the plasma membrane localization of PYL4.(6)StLTP10 positively regulates ABA signaling.Previous studies have reported that the membrane-localized ABA receptors play important roles in early ABA signaling perception and transduction.To test whether StLTP10 has a influence on ABA signaling pathway by regulating PYL4,the expression of several downstream genes of PYL4,such as ABI3,ABI5,RAB18,etc.in WT and transgenic plants were examined by using qRT-PCR.It was found that StLTP10 overexpression can significantly enhance the expression of those genes.Additionally,ABA-mediated root growth inhibition was more obvious in StLTP10-OE lines than that in WT,indicating that StLTP10 positively regulates ABA signaling.(7)StLTP10 cooperates with PYL4 to regulate stomatal closure after pathogen infection.At the early stage of pathogen infection,the stomatal closure induced by ABA plays a key role in preventing pathogen invasion.To explore whether StLTP10 could participate in ABA-induced stomatal movement,the stomatal behavior of WT and transgenic plants leaves were examined after treated with ABA.The results showed that the stomatal closure of StLTP10-OE plants leaves exhibited more sensitive to ABA compared with WT.On the contrary,the RNAi plants leaves showed impaired stomatal closure in response to ABA.The results demonstrate that StLTP10 plays positive role in ABA-induced stomatal closure.To determine whether StLTP10 is also required for stomatal closure during pathogen attack,those leaves were incubated with pathogens and then investigated their stomatal response.The data revealed that the stomatal closure was more susceptible in StLTP10-OE plants compared with that in WT after inoculation,however,the stomatal closure of RNAi plants showed disrupted,implying that StLTP10 is involved in regulating stomatal closure during pathogen attack.To explore the role of PYL4 in stomatal closure,PYL4 silencing lines were generated by using VIGS in potato.It was found that the stomatal closure after P.infestans infection in TRV2::PYL4 was impaired similar to that in StLTP10-RNAi plants.Furthermore,the stomatal phenotype showed no differences between double downregulation lines and single downregulation lines either in normal conditions or P.infestans infection.All together,we conclude that the StLTP10 can cooperate with PYL4 to positively regulate stomatal closure after pathogen infection.(8)WIPK phosphorylates StLTP10 and maintains its protein stability.To further identify the core components of StLTP10 involved in plant immunity against P.infestans,the experiment of screening Y2 H library composed of cDNA from potato leaves was tried again.Finally,the wound-induced protein kinase(WIPK),which is a homologue of well-known MPK3 required for both development and biotic stress response,was of our interest for further study.Disease resistance analysis showed that WIPK can improve the resistance ability of StLTP10.In vitro kinase experiments indicated that WIPK can directly phosphorylate StLTP10.In addition,WIPK can inhibit the degradation of StLTP10 by using in vitro and in vivo protein degradation experiments.In conclusion,WIPK is required for the protein stabilization of StLTP10 through phosphorylation and thus participating in plant disease resistance.In summary,overexpression of StLTP10 reduces the accumulation of ROS though up-regulating ROS scavenging-related genes and promotes the expression of defense-related genes,thus enhancing the plant resistance to P.infestans.Intriguingly,we found that StLTP10 interacts with the ABA receptor PYL4 and MAPK cascade member WIPK.On the one hand,StLTP10 can effect the plasma membrane localization of PYL4,thus positively regulate ABA signaling.Further researches show that StLTP10 cooperates with PYL4 to positively regulate the stomatal closure of leaves during pathogen attack.On the other hand,WIPK can phosphorylate StLTP10 and regulate its protein stability,thus enhancing the disease resistance of StLTP10.Therefore,we uncovered a novel disease resistance pathway in potato which firstly integrates nsLTP with MAPK pathway and ABA signaling.