Functional Analysis Of VmRCO-3 Gene In Valsa Mali

Apple valsa canker is a branch disease caused by Valsa mali,which is known as’apple tree cancer’ and seriously threatens the sustainable development of apple-related industries in China.Previously,it was generally believed that low temperature reduced the resistance of apple trees to pathogens by delaying the healing rate of fruit tree wounds,making them more susceptible to infection.Previous studies in the laboratory have found that low temperature induction can improve the pathogenicity of Valsa mali,but its regulatory mechanism is still unclear.In order to clarify this problem,the research group used the ATMT mutant library to screen a low temperature sensitive mutant,which was named ΔVm86.PCR and southern blot proved that the mutant was a single T-DNA insertion mutant.The right-wing sequence of the mutant gene was cloned by TAIL-PCR,but its complete gene and function were not yet clear.In order to clarify this problem,this study used Tbtools software to locate the gene in the genome of Vm263,and used bioinformatics,molecular biology and biochemistry to determine the biological function of the gene.At the same time,RNA-Seq technology was used to analyze the molecular regulation mode of VmRCO-3 gene in response to low temperature.The main results are as follows:1.The whole genome sequencing analysis of Vmali strain Vm263.The second-generation sequencing and the third-generation sequencing obtained 6.79 Gb and 6.61 Gb of reads,and the sequencing depth was 161×and 158×,respectively.After the genome assembly of strain Vm263,the size was 42.83 Mb,the GC content was 49.21%,the N50 value was 5.09 Mb,and the number of Contigs was 24.The genome of strain Vm263 was annotated to obtain 1291 CAZy-encoding proteins,2519 genes were annotated in the PHI database,and 691 secretory proteins were predicted based on the presence or absence of N-terminal signal peptides and transmembrane domains.2.Cloning and bioinformatics analysis of mutant gene.The nucleotide sequence of the mutant gene was obtained by comparing the mutant gene with the Vm263 genome by TBtools software.The gene was 1822 bp in length,containing a 124 bp intron,encoding 566 amino acids,no signal peptide,containing a transmembrane domain,and subcellular localization on the cell membrane.Amino acid sequence alignment showed that the conserved domain of the mutant gene was MFS_HXT.Phylogenetic analysis showed that the amino acid sequence of the mutant gene had high homology with RCO-3 protein,so it was named VmRCO-3.3.Gene knockout and gene complementation of VmRCO-3.The knockout vector and complement vector of VmRCO-3 gene were constructed by seamless cloning.The knockout vector was transferred into the protoplast of WT strain by PEG-CaCl2 mediated protoplast transformation method,and the complement vector was transferred into the protoplast of △VmRCO-3.One △ VmRCO-3 and one △VmRCO-3-C were obtained by PCR and qPCR.4.Biological function analysis of VmRCO-3.WT,△VmRCO-3 and △VmRCO-3-C strains were determined for colony and mycelium morphology,mycelial growth rate,conidial germination rate,abiotic factor stress,growth of different carbon source medium and pathogenicity.The results showed that WT,△ VmRCO-3 and △VmRCO-3-C had no difference in the morphology and color of the colony on the front and back of the colony.However,compared with WT,△ VmRCO-3 had shorter bifurcation of mycelium,significantly faster growth rate,higher conidia germination rate,higher sucrose absorption and utilization rate,and significantly higher pathogenicity.At the same time,VmRCO-3 gene was involved in cell wall osmotic stress and oxidative stress.Under 0.02%SDS and 9 mol/L H2O2 conditions,the mycelial growth inhibition rate of knockout mutant △ VmRCO-3 was 7.49%and 5.56%higher than that of WT,respectively.After gene complementation,it returned to the original level similar to the wild type.After low temperature treatment,the mycelial growth rate of WT was significantly accelerated,the conidia germination rate was significantly decreased,and the pathogenicity was significantly improved.Compared with WT,the growth rate of △ VmRCO-3 mycelium after low temperature treatment decreased significantly,the inhibition rate of conidia germination decreased significantly,and the growth rate of pathogenicity decreased significantly.5.Transcriptome analysis of VmRCO-3 gene deletion mutants.We performed RNA extraction and RNA-seq sequencing analysis of WT and △ VmRCO-3 mycelia treated at 25℃ and 5℃.Bioinformatics analysis showed that there were 2876 differentially expressed genes in the wild-type mycelium of V.mali after low temperature induction,of which 1261 genes were relatively up-regulated and 1615 genes were relatively down-regulated.Compared with WT,△ VmRCO-3 had 499 differentially expressed genes,of which 332 genes were relatively up-regulated and 177 genes were relatively down-regulated.Ten genes were selected from the transcriptome for qRT-PCR validation of RNA-seq data.The results showed that the qRT-PCR expression trend of 10 genes was the same as that of RNA-seq,indicating that the transcriptome sequencing data was reliable.GO enrichment analysis showed that after VmRCO-3 gene knockout,DEGs were significantly enriched in transmembrane transporter activity,transporter activity,integral membrane components,lipid catabolic process and other pathways.KEGG PATHWAY functional enrichment analysis showed that after VmRCO-3 gene knockout,differentially expressed genes were significantly enriched in fructose and mannose metabolism,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolites,nucleotide sugar biosynthesis,pentose phosphate pathway and other pathways.Through Venn analysis,VmRCO-3 regulated the up-regulated expression of five genes annotated in the PHI database as enhancing pathogenicity.In summary,the VmRCO-3 gene of Vmali is a key gene that can respond to low temperature and plays an important role in the regulation of pathogenicity.It can affect mycelial morphology,mycelial growth rate,conidia germination rate,participate in cell wall osmotic stress and oxidative stress,and affect sugar metabolism.This study preliminarily clarified the function of VmRCO-3 gene and laid a foundation for further research.