| Potato late blight caused by Phytophthora infestans is one of the most devastating diseases in agricultural system,threatening the global production security of potatoes and tomatoes.Due to the rapid adaptative feature of this aggressive plant pathogen,it is difficult to effectively reduce the agricultural loss caused by P.infestans through growing the current resistant cultivars.Therefore,broad-spectrum and durable Resistance genes to P.infestans(Rpi)need to be further explored and utilized.The immune function of resistance receptors encoding by plant resistance genes rely on their specific recognition of the Phytophthora avirulence(AVR).Dissecting the mode of actions of how plant Rpi receptors recognize pathogen AVR effectors will greatly help to accurately design and improve crop resistance,providing solutions for safe and efficient crop production.The potato disease resistance gene Rpi-vnt1.1 from wild potato species(Solanum venturi)encodes a typical NLR(Nucleotide-binding Leucine-rich Repeat)receptor,and recognize AVRvntl,a conserved AVR protein,to confer broad-spectrum resistance against most of of P.infestans filed isolates.The cultivated potato cultivars carrying Rpi-vnt1.1 are available in market.However,the molecular basis of how Rpi-vnt1.1 recognize AVRvntl reamins unclear,which restricts the further utilization of this broad-spectrum resistance gene.This study focused on the potato resistant mechanism mediated by NLR receptor Rpi-vnt1.1,and conducted research using materials such as potato and model plant Nicotiana benthamiana.Our preliminary observation that chloroplast glycerate kinase(GLYK)is the interact protein of AVRvntl and contributes to Rpi-vnt1.1 resistance provide key clue for this study.Our study here described a systematic and in-depth analysis of the molecular mechanism how Rpi-vnt1.1 confer potato resistance to P.infestans in a AVRvntl specific manner:Plant chloroplast protein GLYK was a key game player in Rpi-vnt1.1 mediated immune response.In order to dissecting the mode of actions Rpi-vnt1.1 mediates immune response,we identified GLYK as one of AVRvntl binding proteins through yeast two-hybrid(Y2H)screening.Y2H,in vitro and in vivo immunoprecipitation experiments demonstrated that AVRvntl interacts with GLYK,and the interaction require a full sequence of the chloroplast transit peptide(cTP)of GLYK.Virus induced gene silencing(VIGS)and complementation of synthetic(Syn)GLYK sequences assays in N.benthamiana collectively confirmed that GLYK is specifically requiredfor Rpi-vnt1.1 mediated immune response.In this study,an RNAi interference vector was introduced into the DesireeRpi-vnt1.1potato to trigger stable silencing of GLYK gene.It was found that the GLYK silenced potato lines still express Rpi-vnt1.1,but their resistance against P.infestans isolates carrying AVRvntl was lost to different degrees.The above results validated that GLYK is a key factor for Rpi-vnt1.1 immune function,providing a genetic frame work to further study the molecular mechanism of how Rpi-vnt1.1-AVRvntl interaction.The alternative promoter selection(APS)regulated by light conditions occurs in potato GLYK gene locus was,leding to a light-dependent resistance conferred by Rpi-vnt1.1.In this study,we found that Rpi-vnt1.1 conferred resistance to late blight depends on light and was significantly impaired in dark.The transcription of AtGLYK,potato GLYK orthologous gene,is regulated by light-dependent APS in Arabidopsis.We therefore speculated that Rpi-vnt1.1 mediated light-dependent resistance links to APS in GLYK locus.In this study,5'RACE and qRT-PCR assays were used to validate that APS exists in potato GLYK gene locus under different light conditions,yielding two major transcript isoforms namely StGLYKFL and StGLYKcyt.StGLYKFL is the dominant transcript under light condition,and its translation product StGLYKFL are located and functions in chloroplast,while the isoform StGLYKcyt accumulates in dark condition with its product locates in cytoplasm and participates in the photorespiratory bypass.In vivo and in vitro assays found that StGLYKcyt are not able to be targeted by AVRvntl due to the loss of cTP domain.The genetic complementation assays on NbGLYK silenced plants validated that StGLYKcyt does not restore the Rpi-vnt1.1 immune function in GLYK silenced plants.In summary,these results revealed that the APS regulation of GLYK is one of the major mechanisms of light-dependent immune response conferred by Rpi-vnt1.1.The immune function of Rpi-vnt1.1 depends on the physical interaction between AVRvntl and GLYK.In order to further explore the molecular mechanism how Rpi-vnt1.1 recognize AVRvntl,we examined the three-dimensional crystal structure of AVRvnt1 harvested from our collaborator.AVRvnt1DE,a C-termina1 negatively charged amino acid mutant,does not interact with GLYK and fails to trigger HR on N.benthamiana.The in vivo AVRvntl/GLYK complex was further co-immunoprecipitated and submitted for Liquid chromatography-tandem mass spectrometry(LC-MS/MS)detection.The peptides from the LRR domain of the Rpi-vnt1.1 were consistently identified.Further Co-immunoprecipitation assay verified that LRR domain of the Rpi-vnt1.1(Rpi-vnt1.1LRR)interacts with GLYK and AVRvnt1,but it fails to interact with AVRvnt1DE,suggesting that AVRvntl/GLYK complex is necessary for Rpi-vnt1.1 activated immune function.In order to screen natural AVRvntl allele that evade Rpi-vnt1.1 recognition,we harvested 8 AVRvntl allelic sequences from P.infestans and its sister species.The HR assays found that PipAVRvntl from Phytophthora ipomoeae completely escapes Rpi-vnt1.1 recognition.The interaction experiments such as Y2H,in vitro pull-down and in vivo Co-IP,it was found that PipAVRvntl still binds GLYK,suggesting that in additional to AVRvntl/GLYK interaction,the biochemical features or and conformation of AVRvntl/GLYK complex is required for Rpi-vnt1.1 immune function.These results not only provide insight into the activation mechanism of Rpi-vnt1.1,but also spark research clues for the future research on the mode of actions of.AVRvntl promotes GLYK degradation leading to increased host susceptibility.In this study,we found that silencing of the GLYK in potato and N.benthamiana promote plant susceptibility to P.infestans.Complementation of synthetic functional StGLYK instead of the kinase dead mutant StGLYKK222R rescured the basal immunity in N.benthamiana,indicating that GLYK is a positive immune regulator in a kinase activity dependent manner.Cell biology observations and western blot detections revealed that AVRvntl expression in planta or inoculation with P.infestans significantly abolished StGLYK accumulation in chloroplast.Whereas pretreatment with 26S proteasome inhibitor Bortezomib in N.benthamiana leaves increased StGLYK accumulation in the cytoplasm,indicating that AVRvntl blocked StGLYK uptake into chloroplast and promote its cytosolic degradation by 26S proteasome.We used transcriptome sequencing(RNA-seq)analysis revealed that the expression of PR genes PR1,ERF4 and hormone-related genes GH3,EBF1 in GLYK silenced potato at 1 dpi and 2 dpi were significantly down-regulated.These result reveals a novel mechanism that P.infestans effector protein interferes with chloroplast function and consequently impairs plant immune responses.In summary,this thesis systematically clarified the mode of actions of how potato disease resistance receptor Rpi-vnt1.1 recognition P.infestans AVRvntl to activate plant immunity in a light dependent manner,providing both theoretical frame concept and practical guidance for developing and utilizing potato Rpi-vnt1.l gene,and as such to contribute the sustainable and efficient disease management for potato late blight. |
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