Characterization Of Avirulence Genes On Nonhost Plant And Molecular Modification Of Resistance Gene For Phytophthora. Infestans

Potato late blight caused by Phytophthora infestans is one of the devastating diseases.Breeding resistant varieties have become one of the major goals of potato production.P.infestans contains the largest genome among oomycetes which encoding more than 500RXLR effectors.During the infection,they help to create a suitable environment for invasion and proliferation by inhibiting host defense response or hijacking the plant susceptible factors.In this study,I have done two parts of researches,for characterization of avirulence genes on Solanum nigrum,a nonhost plant for P.infestans,and molecular modification of resistance gene in vitro.The main results were described as followed:1.A transient expression gene pool containing 251 RXLR effectors was constructed cooperatively by using the template genomic DNA of HLJ strain,a A1 anastomosis group isolate of P.infestans.After transiently expressed on Solanum nigrum SN022,PITG₁6245.2^HLJ,PITG₂0301.2 and PITG₂0301.2^HLJ were identified as the candidate avirulence genes which could specifically induce the hypersensitive response?HR?.?1?PITG₁6245.2^HLJ.PITG₁6245.2^HLJ is significantly up-regulated during the P.infestans infection,especially in early biotrophic stage.Overexpressing of PITG₁6245.2^HLJ in Solanum tuberosum and Nicotiana benthamiana resulted in down-regulating the expression level of the defense-related genes,affecting the plant growth,and promoting the colonization of P.infestans in the host,indicating that PITG₁6245.2 is an important virulent factor.Homologous sequences of PITG₁6245.2 were found in all 11 isolates of P.infestans.Six out of 11 homologous sequences have been identified to lost HR activating ability on SN022.Combined with the site-directed mutagenesis,the amino acids at the positions 53,66,70 and78 were identified as the key sites for activating HR on SN022.After screening the cDNA library through yeast two-hybrid?Y2H?with PITG₁6245.2^HLJLJ as a bait,StPR4b is identified to an interactor and a positive regulator of plant immunity.Coexpression with StPR4b,PITG₁6245.2^HLJ was prevent to enter into nucleus.Besides,StPR4b would accumulate on cytomembrane from cytoplasm to enhance the host resistance to P.infestans.?2?PITG₂0301.2.PTIG₂0301.2 belongs to Avrblb2 family that is down-regulated after inoculating the P.infestans on potato.They are existed in 14 Phytophthoras isolates which present as six alleles and share over 90%amino acid identity.Two out of six alleles can escape the detection of S.nigrum.Combined with site-directed mutagenesis it was found that the forty-second amino acid is the key site for activating HR on SN022.Overexpression of PITG₂0301.2^HLJ in two hosts down-regulate the expression level of defense-related genes,promote the colonization of P.infestans and affect the normal growth.PITG₂0301.2^HLJ also inhibited the HR induced by NIP,Avh238 and Avh241 in N.benthamiana,suggesting that PITG₂0301.2^HLJ could inhibit both the PTI and ETI.?3?PITG₀4194.1.PITG₀4194.1 had4 kinds of homologous sequence among on total 14 Phytophthoras isolates.All four alleles could activate HR in S.nigrum SN022.PITG₀4194.1^HLJ could also down-regulate the expression of defense-related genes and promote the colonization of P.infestans in potatoes.PITG₀4194.1^HLJ could suppress the PTI and ETI by inhibiting the HR induced by INF1,BAX,NIP,Avh238 and Avh241 in N.benthamiana.2.To investigate how PR4b affects the recognition between the candidate Avr genes and S.nigrum,the sequence of PR4b in S.nigrum and S.tuberosum was analysed.There was only one amino acid difference between SnPR4b and StPR4b which has no negative effect on the interaction with candidate Avr.Interestingly,PITG₁6245.2^HLJ,PTIG₀4194.1^HLJ and PTIG₂0301.2^HLJ could directly interact with SnPR4b.Silencing SnPR4b through the VIGS could inhibit the HR induced by three candidate Avr genes in SN022,indicated that SnPR4b is a key gene for non-host to recognize effectors.However,SN008,another S.nigrum collections,could not recongize any of three candidate Avr genes without triggered HR after transiently expression.The sequence of SnPR4b was identical the same in SN022 and SN008.Yet,SnPR4b is localized to the cytomembrane in SN022 and cytoplasm in SN008.In addition,the expression level of SnPR4b is significantly higher in SN022 than in SN0008.This might be the key mechanism for SnPR4b to identify candidate Avr genes.3.SnPR4b can interact with SnLRR1 directly.Silencing the SnLRR1 gene can reduce the HR induced by PITG₁62545.2^HLJ in S.nigrum,while the HR induced by PITG₂0301.2^HLJ and PITG₀4194.1^HLJ was not affected.We co-expressed three candidate Avr genes with SnPR4b and SnLRR1 in N.benthamiana.Only the HR induced by PITG₁6245.2 was restored.These results suggest that SnLRR1 is one of the downstream genes of SnPR4b,which participates in the pathway of identifying candidate Avr genes in S.nigrum.The interaction between SnPR4b and SnLRR1^SN008 was not effected by the two amino acid loci differences in SnLRR1^SN022 and SnLRR1^SN008.However,the expression level of SnLRR1 in SN022 was significantly higher than in SN0008.4.The disease resistance gene that specifically recognizes RXLR effectors is the key to breeding resistant varieties against late blight.Most of the disease-resistant genes have been cloned by traditional gene cloning methods which were time-consuming and laborious.The R genes usually mediate resistance to a few isolates by identifying 1-2 RXLR effectors.The resistance spectrum is narrow and easy to be overcome by pathogen evolution.Based on the above mechanism that SnPR4b and SnLRR1 altered resistance recognition by weakly enhancing transcriptional expression,we tried to change the expression strategy of cloned resistance gene to modify a new resistance gene.Rpi-mcq1.2 is a R gene with partial resistance to only a few P.infestans isolates.The Rpi-Vnt1.1 promoter was fused to the CC-NB-LRR region of the Rpi-mcq1.2 to form a new potato late blight R gene Rpi-OM1.2.Compared with Rpi-mcq1.2,the expression level of Rpi-OM1.2 in transgenic potatoes increased about 1.5 times.Stable expression of Rpi-OM1.2 did not affect the economical character of potatoes.However,the resistance of Rpi-OM1.2 to potato late blight wad significantly enhanced,and the resistance spectrum was significantly broadened.In order to explore the mechanism of disease resistance,co-expression Rpi-OM1.2 and RXLR genes of transient expression gene pool in N.benthamiana.Six effectors were screened with no obvious homology,which was induced HR by recognizing of Rpi-OM1.2.Howere,Rpi-mcq1.2 can only identify two of them.This would be the basis of broad-spectrum disease resistance.In conclusion,this study identified three RXLR effectors that specifically induce HR in non-hostS.nigrum.Theinteractionrecognitionmechanismof PITG₁6245.2-SnPR4b-SnLRR1 was analyzed,which provides a direction for the new application of RXLR effectors.At the same time,through genetic modification to increase the expression of weak resistance genes,a new resistance genes with strong resistance,broad resistance spectrum and no adverse effects were successfully created.It has not been reported before,which provides new ideas for the prevention of late blight and the breeding of new resistant varieties.