Evaluation Of Resistance To PVX In Major Potato Cultivars In Northeast China

Potato is the fourth largest crops in the world and is also a new main-grain promoted by China,which plays an important role in China’s economy.Although China is the largest potato tube producer for many years,the average yield of potato tube of China is lower than most countries.One important reason is long term of vegetative propagation leads to the accumulation of viruses on potato tubes.Potato virus X(PVX)is one of serious viral threats to potato industry in China.Although PVX infection alone does not induce severe symptoms in many potato cultivars,co-infection of other viruses may cause on serious damages to potato yield and quality.Heilongjiang province is one of the main potato-producing regions in China.Identification the resistance of main potato cultivars can help to improve yield by planning potato variety pattern.In addition,understand the mechanisms of PVX resistance can establish the foundation for potato resistance breeding.In this study,the PVX resistance of 23 main potato cultivars in northeast China,e.g.,Kexin-13,Eugene,Atlantic,etc,was studied Kexin-1 and their mechanisms of resistance was analyzed.Results show that the resistance of all 4 resistant potato varieties is conferred by Rx gene and found a new resistance mechanism in Waiyin-1.The main results are listed as follows:1.PVX resistances of main potato cultivars in northeast China were studied:Virus-free tissue culture seedling of 23 cultivars were inoculated with PVX by agroinfiltration.Systematic leaves were tested by DAS-ELISA and RT-PCR at 12 days postinoculation(dpi).Results show that 5 out of the 23 potato cultivars,namely Longshu 3,Eugene,Atlantic,Waiyin-1,and Waiyin 2,are resistant to PVX.A yellow fluorescence protein(YFP)-tagged PVX infectious clone(pGR107-YFP)was constructed and used to confirm the resistance.Results confirm that Longshu 3,Eugene,Atlantic,and Waiyin 2 are immune to PVX infection,whereas Waiyin-1 delays PVX infection,indicating that Waiyin-1 has a different resistant mechanism to other four cultivars.2.Identification of Rx gene in resistant cultivars:Presence of Rx gene in the five resistant cultivars was identified by RT-PCR using primers designed according to conserved nucleotide sequence of Rx gene.A specific band with expected size was amplified in Longshu 3,Eugene,Atlantic,and Waiyin 2,while no corresponding band was amplified in Waiyin-1.Amplified fragments were cloned and sequenced,and results demonstrate that amplified fragements are the nucleotide sequences of Rx gene.Moreover,nucleotide sequences of Rx gene from Atlantic and Longshu 3 were identical to that of Rx1,whereas nucleotide sequences of Rx gene from Eugene and Waiyin 2 are Rx homologs.These results indicate that Longshu 3,Eugene,Atlantic,and Waiyin 2 have Rx-mediated extreme resistance to PVX,while the resistance mechanism of Waiyin-1 was unknown.3.The resistance mechanism of Waiyin-1 was explored:Waiyin-1 and Kexin-1,which as used as a susceptible control,were mechanically inoculated with pGR107-YFP,and the infection of PVX was recorded by monitering the spread of YFP.YFP fluorescence was observed on the systemic leaves of Kexin-lat 10 dpi,whereas YFP fluorescence was not observed on the systemic leaves of Waiyin-1 until 14 dpi.Accumulations of PVX genomic RNA were evaluated by quantitative reverse transcription-polymerase chain reaction(qRT-RCR)with primers designed according to PVX Coat Protein(CP)gene.Results show that PVX genome accumulated rapidly in Kexin-1 from 48-96 hours post-inoculation(hpi),but increased slowly in Waiyin-1 of the same time period,indicating that replication or movement of PVX may be inhibited in Waiyin-1.Analyzing fluorescence intensity of PVX-infected single cells showed that the YFP fluorescence in Waiyin-1 was much weaker than that in Kexin-1 at the same time point,suggesting that the replication of PVX was inhibited in Waiyin-1.Moreover,a movement defective PVX infectious clone(pGR107-TGBp2Δ52-60-YFP)with was constructed and mechanically inoculated on Kexin-1 and Waiyin-1.Accumulations of PVX genomic RNA were evaluated by qRT-PCR.Results showed that PVX genome in Waiyin-1 accumulated slower than in Kexin-1 at the same time points,indicating that Waiyin-1 inhibits PVX replication.In addition,the expression of PR genes in Waiyin-1 was higher than that in Kexin-1,suggesting that the antiviral immunity has been activated after PVX inoculation in Waiyin-1.