Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

Characterization of NPR1 and NPR4 genes from mulberry (Morus multicaulis) and their roles in development and stress resistance.

The quality and quantity of mulberry leaves are often affected by various environmental factors. The plant NPR1 and its homologous genes are important for plant systemic acquired resistance. Here, the full-length cDNAs encoding the NPR1 and NPR4 genes (designated MuNPR1 and MuNPR4, respectively) were isolated from Morus multicaulis. Sequence analysis of the amino acids and protein modeling of the MuNPR1 and MuNPR4 proteins showed that MuNPR1 shares some conserved characteristics with its homolog MuNPR4. MuNPR1 was shown to have different expression patterns than MuNPR4 in mulberry plants. Interestingly, MuNPR1 or MuNPR4 transgenic Arabidopsis produced an early flowering phenotype, and the expression of the pathogenesis-related 1a gene was promoted in MuNPR1 transgenic Arabidopsis. The MuNPR1 transgenic plants showed more resistance to Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000) than did the wild-type Arabidopsis. Moreover, the ectopic expression of MuNPR1 might lead to enhanced scavenging ability and suppress collase accumulation. In contrast, the MuNPR4 transgenic Arabidopsis were hypersensitive to Pst. DC3000 infection. In addition, transgenic Arabidopsis with the ectopic expression of either MuNPR1 or MuNPR4 showed sensitivity to salt and drought stresses. Our data suggest that both the MuNPR1 and MuNPR4 genes play a role in the coordination between signaling pathways, and the information provided here enables the in-depth functional analysis of the MuNPR1 and MuNPR4 genes and may promote mulberry resistance breeding in the future.

A Novel LncRNA, MuLnc1, Associated With Environmental Stress in Mulberry (Morus multicaulis).

Environmental stresses are major constraints that limit the leaf productivity and quality of mulberry. LncRNAs have emerged as important regulators in response to biotic and abiotic stresses in plants. However, the functions and mechanisms of most lncRNAs remain largely unknown. A novel lncRNA designated as MuLnc1 was found to be cleaved by mul-miR3954 and produce secondary siRNAs in a 21 nt phase in mulberry. It was demonstrated that one of the siRNAs produced, si161579, can silence the expression of the calmodulin-like protein gene CML27 of mulberry (MuCML27). When MuCML27 was heterologously expressed in Arabidopsis, the transgenic plants exhibited enhanced resistance to Botrytis cinerea and Pseudomonas syringae pv tomato DC3000. In addition, the transgenic MuCML27-overexpressing Arabidopsis plants are more tolerant to salt and drought stresses. Furthermore, the network of mul-miR3954-MuLnc1-siRNAs-mRNAs was modeled to elucidate the interaction between lncRNAs and sRNAs with mRNAs. All of these, taken together, suggest that MuLnc1 was associated with environmental stress in mulberry and may be considered as a potential genetic improvement target gene of mulberry. The information provided may shed light on the complicated gene expression regulatory mechanisms in mulberry stress responses.

MiRNA-seq-based profiles of miRNAs in mulberry phloem sap provide insight into the pathogenic mechanisms of mulberry yellow dwarf disease.

A wide range of miRNAs have been identified as phloem-mobile molecules that play important roles in coordinating plant development and physiology. Phytoplasmas are associated with hundreds of plant diseases, and the pathogenesis involved in the interactions between phytoplasmas and plants is still poorly understood. To analyse the molecular mechanisms of phytoplasma pathogenicity, the miRNAs profiles in mulberry phloem saps were examined in response to phytoplasma infection. A total of 86 conserved miRNAs and 19 novel miRNAs were identified, and 30 conserved miRNAs and 13 novel miRNAs were differentially expressed upon infection with phytoplasmas. The target genes of the differentially expressed miRNAs are involved in diverse signalling pathways showing the complex interactions between mulberry and phytoplasma. Interestingly, we found that mul-miR482a-5p was up-regulated in the infected phloem saps, and grafting experiments showed that it can be transported from scions to rootstock. Based on the results, the complexity and roles of the miRNAs in phloem sap and the potential molecular mechanisms of their changes were discussed. It is likely that the phytoplasma-responsive miRNAs in the phloem sap modulate multiple pathways and work cooperatively in response to phytoplasma infection, and their expression changes may be responsible for some symptoms in the infected plants.

The Latex Protein MLX56 from Mulberry (Morus multicaulis) Protects Plants against Insect Pests and Pathogens.

Biotic stresses are major constraints limiting the leaf quality and productivity of mulberry. MLX56 is a unique chitin-binding protein isolated from Shin-Ichinose (Morus alba) latex that displays toxicity against lepidopteran caterpillars. In this study, the full-length cDNA encoding MLX56 was isolated from Husang 32 (M. multicaulis) and designated HMLX56. Amino acid sequence analysis and protein modeling of three MLX56 proteins showed that they were highly conserved among Morus species. Tissue expression pattern analysis showed that the HMLX56 gene was strongly expressed in mulberry bark and leaves but only slightly expressed in fruits. In addition, analysis of GUS expression indicated that the promoter of HMLX56 showed higher transcriptional activity along the vascular strands, and its activity can be regulated by various environmental factors. Like the MLX56 protein from M. alba, the HMLX56 protein showed toxicity to Plutella xylostella. Moreover, when the HMLX56 gene was ectopically expressed in Arabidopsis, the transgenic plants showed enhanced resistance to aphids, the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our data suggest that the HMLX56 protein has a lectin-like molecular structure consisting of two hevein-like chitin-binding domains which provide not only chitin-binding activities but also other mechanisms of defense. The information provided here improves our understanding of the potential functions and defense mechanisms of MLX56 proteins, enabling in-depth functional analysis of latex exudates and perhaps facilitating mulberry genetic improvement in the future.