Study On The Molecular And Physiological Change On The Pinus Massoniana To Bursaphelenchus Xylophilus Infestation

Pine wilt disease(PWD)is a devastating disease affecting the growth of pine,often leading to withering and death.To reveal the underlying mechanism of the phenomenons,Pinus massoniana was used as a donor plant to explore the miRNA and mRNA expression pattern from the pine seedling underwent Bursaphelenchus xylophilus early infestation with different days’ duration,and the high-throughput sequencing was taken to explore these miRNA and mRNA expression.Based on the results,comparison of the transcriptional change between the treatment groups and the corresponding control group was conducted,the differentially expressed genes(DEGs)and their enriched pathway were also indicated,with the aims to reveal the vital DEGs involved in the regulation of plant resistance to the disease.The change in major metabolic pathways and physiological resistance,in which the miRNA target genes or DEGs participated were assessed.Basing on the above studies,the molecular and physiological mechanism on P.massoniana in response to B.xylophilus infestation was indicated.The main results are given as follows,Firstly,the high through-out RNA-Seq was used to study the miRNA expression pattern on the P.massoniana infestation with B.xylophilus for 1,2,and 3 days,as well the corresponding control group,which was infested with sterilizing ddH2O.A comparison between the treatments and control showed that the number of differentially expressed miRNAs increased firstly and then decreased in samples within 3 days of infection,and it was the highest in the sample collected on 2 days after infection.Enrichment analysis of miRNA target genes in pine needles on different days after infection showed that the target genes were mainly enriched in 2 metabolic pathways:the plant hormone signal transduction pathway and the RNA transport pathway.A further comparison of the samples showed that 10 miRNAs exhibited differential expression in P.massoniana on different days after infection.The corresponding target genes were also significantly enriched in pathways such as the plant hormone signaling pathway.It was suggested that the B.xylophilus infestation resulted in change expression of miRNAs mainly involved in the plant hormone signal transduction.The dynamic expression of the concerned miRNAs and their target genes was detected,these genes which including miR5054 and its target gene histidine kinase 4-like,miR384 and its target gene pre-mRNA-splicing factor SPF27,miR5059 and its target gene small nuclear ribonucleoprotein-associated protein,as well as novel-m0012-5p and its target gene protein ethylene insensitive 3-like isoform 1.The determination of these genes expression on the P.massoniana infestation with B.xylophilus was conducted by using quantitative PCR(qPCR).The results showed that 4 miRNAs out of the tested 5 miRNAs were down-regulated in the P.massoniana infestation with B.xylophilus,while the miR5054 was up-regulated on the first day of treatment,and then down-regulated on the second day and finally up-regulated on the third day of infestation,which were compared with the control group.In contrast to the expression pattern of the five miRNAs,the predicted target genes presented the reverse expression pattern to their corresponding miRNA,which validates the negative role of miRNA in the regulation of gene transcript level.Further detection of the concentrations of zeatin(a kind of cytokinin)and indole acetic acid(IAA)in the samples showed that concentrations of these two plant hormones in the needle tissues of treatment groups were both significantly lower than that of the control group within the infestation for 5 days,after that,the concentrations of zeatin and IAA were increased in the B.xylophilus-treated P.massoniana as the day increasing,but there was no significant difference between treatment and control group.At the 14 days of infestation,the concentrations of the zeatin and IAA was decreased and significantly lower in the treatment group than the control group,which suggested the negative influence on the biosynthesis of plant hormones.The above results indicated that B.xylophilus infection resulted in the changing expression of miRNAs related to the plant hormone signaling pathway in P.massoniana,which depresses plant hormone synthesis and ultimately affected the growth of P.massoniana.Meanwhile,the mRNA expression pattern on the P.massoniana infestation with B.xylophilus for 1,2,and 3 days was also studied.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistance protein-like,and the genes encoded nematode resistance protein-like,calcium binding protein,receptor-like protein kinase,and late embryogenesis abundant-like protein.An increasing number of nematode infestation causes a continuous decline in disease resistance gene expression on different tisseues from P.massoniana.In addition,the infestation of B.xylophilus on P.massoniana also resulted in enriched DEGs involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.Moreover,a downregulation of antioxidant-relative genes was observed in the infested samples,which resulted in a decrease of antioxidant enzyme activity.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and the defense was observed in P.massoniana,together with a decrease in the levels of phytoalexin-like secondary substances and the activity of the antioxidant enzyme.Additionally,the correlation between expression pattern on the miRNA and mRNA was analyzed.Fifteen miRNAs out of the library were considered as candidates,which represented statistically significant interactions with twelve mRNA from the results of the RNA-seq.These miRNA-targeted mRNA encodes the disease resistance gene,CC-NBS-LRR resistance-like protein,and etc.The expression pattern between these mRNA and miRNA was the reverse,which validates the transcriptional silence of miRNA on the target mRNA.Based on the results of this study,it was clear that the infestation of B.xylophilus resulted in increased transcript level of the miRNAs target to disease resistance gene(R gene),which causes the downregulation of R gene expression,and therefore reduces the resistance of P.massoniana to B.xylophilus.Meanwhile,the genes that participated in plant growth,phytoalexin synthesis,and genes encode antioxidase were also down-regulated,following by decreasing of IAA,zeatin and phytoalexin concentrations and the activity of many antioxidase.All of the negative effects from B.xylophilus infestation resulted in withering and ultimately death of P.massoniana.