miRNAs are involved in the pollen development during the CMS occurrence in rice. miRNAs are 20-24 nt endogenously expressed small RNAs that play key roles in the regulation of many growth and developmental processes in plants. The knowledge on cytoplasmic male sterility (CMS) regulation by miRNAs in rice is rather limited. In this study, Illumina sequencing was employed to examine the expression profiles of rice anther miRNAs from the CMS line MeixiangA (MxA) and its maintainer line MeixiangB (MxB). A total of 518 known miRNAs and 144 novel miRNAs were identified during rice anther development. Based on the number of sequencing reads, a total of 24 miRNAs were discovered to be differentially expressed between MxA and MxB, and the results were partially validated by qRT-PCR. Among these, 16 miRNAs were decreased and 8 miRNAs were increased in MxA compared with MxB. Target prediction showed that they target genes encoding EF-hand family proteins, F-box domain-containing proteins, MYB transcription factors, PPR-containing proteins and transposons. The expression patterns for targets of osa-miR528, osa-miR5793, osa-miR1432, osa-miR159, osa-miR812d, osa-miR2118c, osa-miR172d and osa-miR5498 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. These targets have previously been reported to be related with pollen development and male sterility, suggesting that miRNAs might act as regulators of CMS occurrence in rice anthers. Furthermore, miRNA editing events were observed. The U → C and U → A editing phenomenon was validated by molecular cloning and sequencing. These findings contribute to our understanding of the roles of miRNAs during anther development and CMS occurrence in rice.

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel-nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo's fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC(50) values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC(50) value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

Mushroom-forming fungi are complex multicellular organisms that form the basis of a large industry, yet, our understanding of the mechanisms of mushroom development and its responses to various stresses remains limited. The winter mushroom (Flammulina filiformis) is cultivated at a large commercial scale in East Asia and is a species with a preference for low temperatures. This study investigated fruiting body development in F. filiformis by comparing transcriptomes of 4 developmental stages, and compared the developmental genes to a 200-genome dataset to identify conserved genes involved in fruiting body development, and examined the response of heat sensitive and-resistant strains to heat stress. Our data revealed widely conserved genes involved in primordium development of F. filiformis, many of which originated before the emergence of the Agaricomycetes, indicating co-option for complex multicellularity during evolution. We also revealed several notable fruiting-specific genes, including the genes with conserved stipe-specific expression patterns and the others which related to sexual development, water absorption, basidium formation and sporulation, among others. Comparative analysis revealed that heat stress induced more genes in the heat resistant strain (M1) than in the heat sensitive one (XR). Of particular importance are the hsp70, hsp90 and fes1 genes, which may facilitate the adjustment to heat stress in the early stages of fruiting body development. These data highlighted novel genes involved in complex multicellular development in fungi and aid further studies on gene function and efforts to improve the productivity and heat tolerance in mushroom-forming fungi.

Most mitochondrial proteins are synthesized in the cytosol as precursor and imported into the mitochondria by Tom complexes (translocase of outer membrane complexes). Knowledge of the binding mechanism between precursor and Tom20 in plants is very limited. Here, computational methods are employed to improve our understanding of the interactions between both molecules. To this end, we model mitochondrial superoxide dismutase precursor (pSOD) in complex with Tom20 in Oryza sativa (OsTom20). In a first stage, five main binding modes were generated using clustering analysis, energy minimization, and expert knowledge. In a second stage, the quality and validity of the resulting complexes is assessed by molecular dynamics (MD) simulations with a generalized Born solvation model. The change in binding free energies is estimated using a computational alanine scanning technique. We identified a particularly favorable complex between pSOD and OsTom20, exhibiting the lowest binding free energy among all candidates and correlating well with experimental data. Furthermore, three independent explicit solvent MD simulations of this structure, each of 100 ns duration, reveal that hydrophobic interactions occur between pSOD and OsTom20, in particular between L158 of pSOD and W81 of OsTom20, as evidenced by analysis of intermolecular distances and corresponding relative free energy landscapes. L158 is part of an interacting LRTLA motif. These results provide new insight into the structural basis and dynamics of precursor recognition by Tom20 in plant, and their generality is supported by sequence alignments with seven other plants.