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. To analyze the molecular mechanisms mediating gene expression in mulberry-phytoplasma interaction, the comprehensive sRNA changes of mulberry leaf in response to phytoplasma-infection were examined. A total of 164 conserved miRNAs and 23 novel miRNAs were identified, and 62 conserved miRNAs and 13 novel miRNAs were found to be involved in the response to phytoplasma-infection. Meanwhile, target genes of the responsive miRNAs were identified by sequencing of the degradome library. In addition, the endogenous siRNAs were sequenced, and their expression profiles were characterized. Interestingly, we found that phytoplasma infection induced the accumulation of mul-miR393-5p which was resulted from the increased transcription of MulMIR393A, and mul-miR393-5p most likely initiate the biogenesis of siRNAs from TIR1 transcript. Based on the results, we can conclude that phytoplasma-responsive sRNAs modulate multiple hormone pathways and play crucial roles in the regulation of development and metabolism. These responsive sRNAs may work cooperatively in the response to phytoplasma-infection and be responsible for some symptoms in the infected plants.

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.

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.

Anthracnose, caused by Colletotrichum dematium, is a serious threat to the production and quality of mulberry leaves in susceptible varieties. Control of the disease has been a major problem in mulberry cultivation. Some strains of Burkholderia cepacia were reported to be useful antagonists of plant pests and could increase the yields of several crop plants. Although B. cepacia Lu10-1 is an endophytic bacterium obtained from mulberry leaves, it has not been deployed to control C. dematium infection in mulberry nor its colonization patterns in mulberry have been studied using GFP reporter or other reporters. The present study sought to evaluate the antifungal and plant-growth-promoting properties of strain Lu10-1, to clarify its specific localization within a mulberry plant, and to better understand its potential as a biocontrol and growth-promoting agent.

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.