Mno-miR164a and MnNAC100 regulate the resistance of mulberry to Botrytis cinerea.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control.

Integrated Analysis of lncRNAs and mRNAs Reveals Complex Gene Network Mediated by lncRNAs and Regulatory Function of MuLRR-RLK-AS in Response to Phytoplasma Infection in Mulberry.

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry.

MnASI1 Mediates Resistance to Botrytis cinerea in Mulberry (Morus notabilis).

Six α-amylase/subtilisin inhibitor genes (MnASIs) were identified from mulberry (Morus notabilis). In this study, bioinformatics and expression pattern analysis of six MnASIs were performed to determine their roles in resistance to B. cinerea. The expression of all six MnASIs was significantly increased under Botrytis cinerea infection. MnASI1, which responded strongly to B. cinerea, was overexpressed in Arabidopsis and mulberry. The resistance of Arabidopsis and mulberry overexpressing MnASI1 gene to B. cinerea was significantly improved, the catalase (CAT) activity was increased, and the malondialdehyde (MDA) content was decreased after inoculation with B. cinerea. At the same time, H2O2 and O2- levels were reduced in MnASI1 transgenic Arabidopsis, reducing the damage of ROS accumulation to plants. In addition, MnASI1 transgenic Arabidopsis increased the expression of the salicylic acid (SA) pathway-related gene AtPR1. This study provides an important reference for further revealing the function of α-amylase/subtilisin inhibitors.

Characterization of GABA-Transaminase Gene from Mulberry (Morus multicaulis) and Its Role in Salt Stress Tolerance.

Gamma-aminobutyric acid (GABA) has been reported to accumulate in plants when subjected to salt stress, and GABA-transaminase (GABA-T) is the main GABA-degrading enzyme in the GABA shunt pathway. So far, the salt tolerance mechanism of the GABA-T gene behind the GABA metabolism remains unclear. In this study, the cDNA (designated MuGABA-T) of GABA-T gene was cloned from mulberry, and our data showed that MuGABA-T protein shares some conserved characteristics with its homologs from several plant species. MuGABA-T gene was constitutively expressed at different levels in mulberry tissues, and was induced substantially by NaCl, ABA and SA. In addition, our results demonstrated that exogenous application of GABA significantly reduced the salt damage index and increased plant resistance to NaCl stress. We further performed a functional analysis of MuGABA-T gene and demonstrated that the content of GABA was reduced in the transgenic MuGABA-T Arabidopsis plants, which accumulated more ROS and exhibited more sensitivity to salt stress than wild-type plants. However, exogenous application of GABA significantly increased the activities of antioxidant enzymes and alleviated the active oxygen-related injury of the transgenic plants under NaCl stress. Moreover, the MuGABA-T gene was overexpressed in the mulberry hairy roots, and similar results were obtained for sensitivity to salt stress in the transgenic mulberry plants. Our results suggest that the MuGABA-T gene plays a pivotal role in GABA catabolism and is responsible for a decrease in salt tolerance, and it may be involved in the ROS pathway in the response to salt stress. Taken together, the information provided here is helpful for further analysis of the function of GABA-T genes, and may promote mulberry resistance breeding in the future.

MicroRNA Profiling During Mulberry (Morus atropurpurea Roxb) Fruit Development and Regulatory Pathway of miR477 for Anthocyanin Accumulation.

To understand the mechanism of small non-coding RNAs (miRNA)-mediated development and ripening of mulberry fruits, three small RNA libraries from mulberry fruits at different development stages were constructed, and 159 conserved miRNAs as well as 86 novel miRNAs were successfully identified. Among the miRNAs identified, there were 90 miRNAs which showed differential expression patterns at different stages of fruit development and ripening. The target genes of these differential expressed (DE) miRNAs were involved in growth and development, transcription and regulation of transcription, metabolic processes, and etc. Interestingly, it was found that the expression level of mul-miR477 was increased with fruit ripening, and it can target the antisense lncRNA (Mul-ABCB19AS) of the ATP binding cassette (ABC) transporter B 19 gene (Mul-ABCB19). Our results showed that mul-miR477 can repress the expression of Mul-ABCB19AS and increase the expression of Mul-ABCB19, and it acted as a positive regulator participating anthocyanin accumulation through the regulatory network of mul-miR477-Mul-ABCB19AS-Mul-ABCB19.

Transcriptome and DNA Methylome Reveal Insights Into Phytoplasma Infection Responses in Mulberry (Morus multicaulis Perr.).

To reveal whether the response of mulberry to phytoplasma infection is associated with genome-wide DNA methylation changes, the methylome and transcriptome patterns of mulberry in response to phytoplasma infection were explored. Though the average methylation level of the infected leaves showed no significant difference from that of healthy leaves, there were 1,253 differentially methylated genes (DMGs) and 1,168 differentially expressed genes (DEGs) in the infected leaves, and 51 genes were found simultaneously to be differently methylated and expressed. It was found that the expression of G-type lectin S-receptor-like serine/threonine protein kinase gene (Mu-GsSRK) was increased, but its methylation level was decreased in the pathogen-infected or salicylic acid (SA)-treated leaves. Overexpression of Mu-GsSRK in Arabidopsis and in the hairy roots of mulberry enhanced transgenic plant resistance to the phytoplasma. Moreover, overexpression of Mu-GsSRK enhanced the expressions of pathogenesis-related protein 1, plant defensin, and cytochrome P450 protein CYP82C2 genes in transgenic plants inoculated with pathogens, which may contribute to the enhanced disease resistance against various pathogens. Finally, the DNA methylation dynamic patterns and functions of the differentially expressed and methylated genes were discussed. The results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection.