Pathogenic function of pectate lyase gene Vmpl4 of Valsa mali in apple

XU Chunjing;SUN Yingchao;WU Yuxing;FENG Hao;GAO Xiaoning;HUANG Lili;College of Plant Protection,Northwest A&F University·State Key Laboratory of Crop Stress Biology for Arid Areas;

Valsa mali;;Pectate lyase;;Gene knockout;;Pathogenicity

【Objective】The Apple Valsa canker, caused by Ascomycete Valsa mali, seriously affects the tree vigor and yield of apple in China. Previous studies inferred that pectinase might play an important role during the pathogenic process of V.mali. Pectate lyase(PL) is a class of pectinase. It cleaves uronic acid into an unsaturated hexenuronic acid and a new reducing end via a β-elimination mechanism. Transcriptome analysis showed that pectate lyase gene Vmpl4 was significantly up-regulated during infection of V.mali. The objective of this study is to verify the pathogenesis-related function of the pectate lyase gene Vmpl4 in V. mali, the utilization of pectin of Vmpl4 deletion mutant and the changes of relative expression levels of other genes in PL family when Vmpl4 was deleted.【Methods】One year old twigs of Malus domestica borkh.‘Fuji'and PDB(Potato Dextrose Broth) medium were inoculated with the wild-type strain 03-8. The lesion bark and the shaking cultured mycelium were collected three days post inoculation as the samples during the infection process and the control. Total RNA of the samples was extracted using the Nucleic acid extraction kit. The relative expression level of Vmpl4 during infection process was detected by quantitive real time PCR(q RT- PCR) with the glucose- 6- phosphate- dehydrogenase(G6PDH) gene as the inner reference gene. Genomic DNA of 03-8 was extracted by CTAB(Hexadecyl tri-methyl ammonium Bromide)method. The upstream and downstream fragments of Vmpl4 were amplified by PCR using the DNA of 03-8 as template. Hygromycin B phosphotransferase gene(hph) was amplified from plasmid p HIG2RHPH2-GFP-GUS. Then Double-joint PCR was used to generate the gene deletion cassette by connecting the three fragments. The transformants were obtained through PEG-mediated protoplast transformation technique, which led to the gene deletion cassette into the protoplasts of 03-8. PCR with four pairs of primers was used to verify the transformants by amplifying the target gene, hph gene, upstream and downstream. A putative deletion mutant had no target gene, but had hph gene, upstream and downstream. Finally, the deletion mutant was detected by Southern bolt using hph gene fragment as hybridization probe. The leaves and twigs of‘Fuji'were inoculated in vitro with the wild-type 03-8 and the deletion mutant,, cultured at 25 ℃ andkept in high humidity, The lesion sizes on leaves and twigs were observed and measured three days and five days respectively after inoculation. The inoculation of03-8 and the deletion mutant were made on PDA and pectin medium, and the vegetable growth of mycelia on PDA was observed two days later, and the colony morphology and sizes on pectin medium were observed and measured three days later. The data of pathogenicity and the colony sizes were analyzed by the statistical software SPSS. The total RNA was extracted from the lesions barks three days after the inoculation. The relative expression levels of other genes in PL family during the process of Vmpl4 deletion mutant infection were performed using q RT-PCR with the sample of 03-8 for comparison.【Results】The results of q RT-PCR showed that, compared with the mycelium of 03-8, Vmpl4 was up-regulated by 10.20 folds at three days post inoculation on apple twigs. The gene deletion cassette was constructed successfully after the amplification of the target gene and hph gene upstream and downstream. Two putative deletion mutant △Vmpl4-7 and △Vmpl4-50 were obtained through PEG-mediated protoplast transformation and PCR detection with four pairs of primers. One deletion mutant △Vmpl4-50 was confirmed as a single locus homologous recombination by Southern blot detection. Data analysis of lesion sizes of 03-8 and △Vmpl4-50 on apple leaves and twigs showed that the pathogenicity of △Vmpl4-50 was significantly reduced by 16.82% and 18.59%, respectively, indicating that Vmpl4 participated in the infection process of V. mali. Compared to 03-8, the colony morphology and sizes of △Vmpl4-50 on the PDA medium showed no change, but the growth rate of △Vmpl4-50 on pectin medium was significantly reduced, which suggested that Vmpl4 might play a role in the degradation of pectin. The deletion of Vmpl4 also caused the expression changes of other genes in PL family. Three genes in PL family were down-regulated, while four genes were up-regulated over 1.5 folds.【Conclusion】Compared with the wild-type 03-8, the pathogenicity of the deletion mutant of Vmpl4 on apple leaves and twigs was significantly reduced, and the growth rate on pectin medium was also reduced. Meanwhile, four genes in the same family with Vmpl4 were significantly up-regulated during the infection process of the deletion mutant of Vmpl4. Therefor, Vmpl4 might participate in pathogenic process of V. mali by degrading pectin, and the other genes in PL family might complemented the pathogenicity when Vmpl4 was deleted, in a anther word, theymight have synergistic effect with Vmpl4 in pathogenicity of V. mali in apple.