Analysis Of The Biological Functions Of The Phytoplasma-responsive Novel MiRNAs In Mulberry

The yellow dwarf disease associated with phytoplasmas is one of the most devastating diseases of mulberry and the pathogenesis involved in the disease is poorly understood. Plant miRNAs recognized as important regulatory factors play very important roles in the regulation of plant growth, signal transduction and environmental response. In this study, three novel miRNAs named as mul-miRn8, mul-miRn26and mul-miRn33which were responsed to phytoplasma infection in mulberry were cloned by PCR technology, and their biological functions were analysised. The results obtained in this study would be particularly useful for understanding the regulatory mechanism of miRNAs in the response to phytoplasma infection, and be aid to reveal the mechanisms underlying of phytoplasma pathogenicity. The main results of this study can be summarized as follows:(1) Expression differences verification of the novel phytoplasma-responsive miRNAs in mulberryBased on the mulberry transcriptome information, the expression differences of the three novel miRNAs between healthy and infected mulberry leaves were verified by Northern blot and qRT-PCR. The results showed that the expression level of mul-miRn8was significantly increased, and both the mul-miRn26and mul-miRn33were decreased significantly in the infected mulberry.(2) Analysis of the expressions and processings of the novel phytoplasma-responsive miRNAs in transgenic ArabidopsisBased on the mulberry transcriptome sequence information, the MulMIRn8, MulMIRn26and MulMIRn33genes were cloned using PCR technology and their plant expression vectors were constructed, and the transgenic Arabidopsis plants of the three genes were obtained successfully. Northern hybridization analysis showed that all miRNAs precursors were processed in the transgenic Arabidopsis plants. Real-time PCR analysis showed that the three genes of mul-miRn8, mul-miRn26and mul-miRn33were able to be processed into mature miRNAs in their transgenic Arabidopsis plants. (3) Analysis of the biological functions of the novel phytoplasma-responsive miRNAsThe transgenic and wild type Arabidopsis plants were exposed to abiotic stresses (drought stress and salt stress) and biotic stress (Pseudomonas syringae pv. tomato DC3000, Pst DC3000infection) to reveal the biological function of the three novel phytoplasma-response miRNAs. The results showed that MulMIRn8transgenic Arabidopsis plants shows strong resistance to Pst DC3000infection and drought stress, however, they shows no significant difference in the resistance to salt stress with wide type Arabidopsis. The data obtained in this study also showed that the MulMIRn26transgenic Arabidopsis plants were more resistant to drought stress and Pst DC3000infection, but more sensitive to salt stress than wild type plants. Although MulMIRn33transgenic Arabidopsis plants shows more resistant to drought and salt stress, they are more sensitive to Pst DC3000infection than wild type plants. Therefore, our results indicated that the biological function of the three novel phytoplasma-response miRNAs were different.(4) Analysis of the biological functions of the target genes of the novel miRNAs responsive to phytoplasma infectionTo further study the mechanism of the three new mul-miRNAs, the target genes of the three novel phytoplasma-response miRNAs were predicted useing bioinformatics techniques. MulBAH1gene, which was homologous to a BAH domain protein, was predicted as one target gene of mul-miRn8. The MulZFl gene annotated as a CCCH domain-containing zinc finger protein was predicted as one target gene of mul-miRn26. One of the target genes of mul-miRn33was predicted as MulDMEl, which is highly homologous to a transcriptional activator (DEMETER). Northern blot analysis indicated that the novel miRNAs obtained form mulberry were expressed in transgenic Arabidopsis, moreover, they can silence their targeted genes in the transgenic plants.The MulBAH1and MulZF1genes were cloned using PCR technology based on the mulberry transcriptome information obtained, and their plant expression vectors were constructed and transformed into Arabidopsis successfully. When the transgenic Arabidopsis were subjected to drought and salt stress and Pst DC3000infection, the transgenic Arabidopsis plants of MulZF1showed more resistance to salt and drought stress and Pst DC3000infection compared with wild type plants. As regards the transgenic Arabidopsi plants of MulBAH1, they showed strong resistance to salt stress but more sensitive to drought stress and pathogen infection than wild type plants.In conclusion, the three miRNAs response to phytoplasma infection not only involved in the response to phytoplasma infection process, but also participate in plant growth and development and a variety of biotic stresses and abiotic stresses processes.