Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri.

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Identification and characterization of the ZRT, IRT-like protein (ZIP) family genes reveal their involvement in growth and kojic acid production in Aspergillus oryzae.

The ZRT, IRT-like protein (ZIP) family exists in many species and plays an important role in many biological processes, but little is known about ZIP genes in Aspergillus oryzae. Here, 10 ZIP genes in A. oryzae were identified and these were classified into four groups based on phylogenetic analysis. The structures of these AoZip genes were determined, which indicated a great divergence of AoZip members from different groups. Synteny analysis revealed that AoZip7, AoZip8, and AoZip10 are conserved among Aspergillus species. We also found that the promoter regions of AoZip2, AoZip7, AoZip8, and AoZip10 contain multiple conserved response elements. Expression analysis revealed that AoZips exhibited different expression patterns in response to different metal treatments. Moreover, overexpression and RNA-interference (RNAi) of AoZip2 led to a decrease in mycelium growth diameter and inhibited conidia formation. AoZip2 overexpression and RNAi strains showed distinct sensitivity to severely Zn/Mn-depleted stress. In addition, kojic acid production was markedly lower in AoZip2 overexpression and RNAi strains than in the control strains, and the expression of kojA, kojR, and kojT was down-regulated in AoZip2 overexpression and RNAi strains. This study provides new insights into our understanding of ZIP genes and lays a foundation for further investigation of their roles in Aspergillus oryzae.