| Development of potato as a staple food is an important initiative to ensure national food security and promote sustainable income for farmers currently.In Inner Mongolia,potato and sunflower are special corps,and play an important role in promoting local economic development.However,the perennial crop rotation of sunflower and potato has made the occurrence and severity of Potato Verticillium Wilt(PVW)increase,this has become a bottleneck for limiting the development of the potato industry in Inner Mongolia region.In this study,we first evaluated resistant level of different potato cultivars against PVW and screened out two varieties which showed dramatically different resistant level as future research materials.Then,systematically infection process of PVW,seeds potato contamination and infection difference between resistance and susceptible variety were studied using GFP tagged V.dahliae.Meanwhile,comparison on expression profiles of both resistant and susceptible potato varieties at different time points were studied via proteomics analysis.The candidate DEPs(Differentially Expressed Proteins)were identified and the corresponding genes were cloned.Finally,the candidate gene function on regulating potato resistance against PVW were studied using VIGS(Virus Induced Gene Silencing)and ATMT mediated transformation system.The results are listed as follows:1.34 potato cultivars were evaluated for resistance to PVW,the result showed that only ‘Zhongshu21’ was identified as highly resistant,accounting for 2.9% of the tested cultivars;11 cultivars including ‘Zhongshu19’ were identified as moderately resistant,accounting for 32.4% of tested cultivars;16 cultivars were identified as moderately susceptible,accounting for 47.1% and 6 cultivars including ‘Xingjia’were identified as highly susceptible,accounting for 17.6% of the cultivars tested.For the level of ‘Zhongshu19’ against V.dahliae is close to high resistance level and virus-free tube seedling is available,both ‘Zhongshu19’ and ‘Xingjia’ were selected as future research materials;2.A Petri dish with filter paper system was established to study the systemic infestation of V.dahliae on potato root;a GFP labeled V.dahliae strain(VdSZW01-08)was obtained by ATMT(Agrobacterium-Mediated Transformation);3.The results of the infestation process of V.dahliae on potato showed that conidia was able to attach to the root hair 2 hpi(hours post inoculation);and start to germinate and form hypha 12 hpi;the hypha were able to invade the intercellular space of the host root system 72 hpi.The hypha penetrated into intercellular space neighboured to the vascular bundle 96 hpi;and it colonized inside vascular bundle at 168 hpi.With time goes on,V.dahliae was able to infected the above-ground tissues through the vascular bundles of the root system and colonized inside the vascular bundle of the tubers through stolons,thus resulting in seed potatoes infection.the germination of the contaminated seed potatoes were able to lead the infection of young sprouts finally.4.The comparison on the infection differences between resistance and susceptible(‘Zhongshu19’ and ‘Xingjia’)were performed with VdSZW01-08.The results showed that there is no difference on the colonization(2 hpi)and germination(12 hpi)of conidia on the root surface of both resistant and susceptible cultivar.24～48 hpi,the hypha started to development on the root surface of both cultivars,but the hypha net on susceptible variety is much more dense than that is on resistant variety.At 72 hpi,strong green signal were observed in the gap of epidermic cell of susceptible,whereas,the green signal was seldom observed on the surface of resistant variety.At 168 hpi: the hypha of V.dahliae could penetrate and colonized inside the periphery of the vascular bundle of the root system of both resistant/susceptible varieties,but,the biomass of V.dahliae inside resistant cultivar is much more less than that is inside susceptible one.This observation is also confirmed by biomass qualification through qRT-PCR using tissues collected from different time points of two tested cultivars.5.Based on the infection process study,we fixed four time points: 0 h,12 h,3 d(72 hpi),7 d(168 hpi),for the sample collection and subject for proteomic analysis.A total of 3 DEPs were screened out by comparison analysis,These DEPs are DHS1(Phospho-2-dehydro-3-deoxyheptonate aldolase),PR1B(Pathogenesis related protein),POD21(Peroxidase).PR1 B was chosen for future function study by validation with qRT-PCR.6.The results of the candidate gene function study evaluated that the disease index of StPR1 B gene silencing plants was 80.0,much higher than that of control 29.5.The qualification of biomass of the hype inside the basal stems of the silenced is 2.8 fold to the control plants.The contamination rate of tubers collected from the silence plant is 80.0%,much higher that that of tuber harvested from the control plants.If we silence the homology gene of StPR1 B in tomato Sl PR1 B,The height and crown of the silence plant were reduced to 22.0% and 45.9% separately compared to the control,respectively.The biomass of hypha colonized in the silenced plants was 1.7 times higher than that of the control plants.These results indicated that the the PR1 B protein has conserved function on the regulation both potato and tomato resistance aganist V.dahliae.7.StPR1 B transgenic lines were generated via ATMT mediated transformation system.Six lines were confirmed positive with the expression level is variable.Three transgenic lines were selected and their resistance was tested via inoculation.The results showed that the disease index of StPR1 B gene over expression plants was 32.12、 32.18 and 29.84,much lower than that of control 51.28;The phenotype of plant wilting,dwarfing,and leaf yellowing was also reduced compared to EV plants.Take together,PR1 B gene positively regulated potato resistance against infection by V.dahliae. |
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