Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication

Comparative proteomic analysis of QTL CTS-12 derived from wild rice (Oryza rufipogon Griff.), in the regulation of cold acclimation and de-acclimation of rice (Oryza sativa L.) in response to severe chilling stress

Abstract Background Rice (Oryza sativa L.) is a thermophilic crop vulnerable to chilling stress. However, common wild rice (Oryza rufipogon Griff.) in Guangxi (China) has the ability to tolerate chilling stress. To better understand the molecular mechanisms underlying chilling tolerance in wild rice, iTRAQ-based proteomic analysis was performed to examine CTS-12, a major chilling tolerance QTL derived from common wild rice, mediated chilling and recovery-induced differentially expressed proteins (DEPs) between the chilling-tolerant rice line DC90 and the chilling-sensitive 9311. Results Comparative analysis identified 206 and 155 DEPs in 9311 and DC90, respectively, in response to the whole period of chilling and recovery. These DEPs were clustered into 6 functional groups in 9311 and 4 in DC90. The majority were enriched in the ‘structural constituent of ribosome’, ‘protein-chromophore linkage’, and ‘photosynthesis and light harvesting’ categories. Short Time-series Expression Miner (STEM) analysis revealed distinct dynamic responses of both chloroplast photosynthetic and ribosomal proteins between 9311 and DC90. Conclusion CTS-12 might mediate the dynamic response of chloroplast photosynthetic and ribosomal proteins in DC90 under chilling (cold acclimation) and recovery (de-acclimation) and thereby enhancing the chilling stress tolerance of this rice line. The identified DEPs and the involvement of CTS-12 in mediating the dynamic response of DC90 at the proteomic level illuminate and deepen the understanding of the mechanisms that underlie chilling stress tolerance in wild rice

An Integration of MicroRNA and Transcriptome Sequencing Analysis Reveal Regulatory Roles of miRNAs in Response to Chilling Stress in Wild Rice

A chromosome single segment substitution line (CSSL) DC90, which was generated by introgressing CTS-12, a locus derived from common wild rice (Oryza rufipogon Griff.), into the 9311 (Oryza sativa L. ssp. indica) background, exhibits a chilling tolerance phenotype under chilling stress. Here, an integration of microRNA (miRNA) deep sequencing and transcriptomic sequencing analysis was performed to explore the expression profiles of miRNAs and their target genes mediated by CTS-12 under chilling stress, and to reveal the possible regulatory mechanisms of miRNAs that are involved in chilling tolerance. Integration analysis revealed that a number of differentially expressed miRNAs (DEMs) and putative target genes with different expression patterns and levels were identified in 9311 and DC90 under chilling stress. KEGG enrichment analysis revealed that the target genes that are regulated by chilling-induced miRNAs are involved in the regulation of various biological processes/pathways, including protein biosynthesis, redox process, photosynthetic process, and chloroplast development in two genotypes. CRISPR/Cas9 editing of the target genes of the key DEMs in a chilling tolerant rice variety Zhonghua 11 (ZH11) found that LOC_Os11g48020 (OsGL1-11), one of the putative target genes of osa-miR1846a/b-5p and encoding a wax synthesis protein, is correlated with a chilling stress tolerance phenotype, implying osa-miR1846a/b-5p/OsGL1-11 plays an important role in CTS-12-mediated chilling stress tolerance regulatory pathway(s). Therefore, we speculate that the CTS-12 may regulate the key miRNA target genes in response to chilling stress by differential regulation of miRNAs in wild rice, thereby resulting in the variation of chilling tolerance phenotype between 9311 and DC90

Genetic analysis and fine mapping of a qualitative trait locus wpb1 for albino panicle branches in rice.

Chloroplast plays an important role in the plant life cycle. However, the details of its development remain elusive in rice. In this study, we report the fine-mapping of a novel rice gene wpb1 (white panicle branch 1), which affects chloroplast biogenesis, from a tropical japonica variety that results in an albino panicle branches at and after the heading stage. The wpb1 variety was crossed with Nipponbare to generate the F2 and BC1F2 populations. Green and white panicle branch phenotypes with a 3:1 segregation ratio was observed in the F2 population. Bulked segregant analysis (BSA) based on whole genome resequencing was conducted to determine the wpb1 locus. A candidate interval spanning from 11.35 to 23.79M (physical position) on chromosome 1 was identified. The results of BSA analysis were verified by a 40K rice SNP-array using the BC1F2 population. A large-scale F2 population was used to pinpoint wpb1, and the locus was further narrowed down to a 95-kb interval. Furthermore, our results showed that the expression levels of the majority of the genes involved in Chl biosynthesis, photosynthesis and chloroplast development were remarkably affected in wpb1 variety and in F2 plants with a white panicle branch phenotype. In line with the results mentioned above, anatomical structural examination and chlorophyll (Chl) content measurement suggested that wpb1 might play an important role in the regulation of chloroplast development. Further cloning and functional characterization of the wpb1 gene will shed light on the molecular mechanism underlying chloroplast development in rice

Effects of Perceptual Learning on Deprivation Amblyopia in Children with Limbal Dermoid: A Randomized Controlled Trial

Limbal dermoid (LD) is a congenital ocular tumor that causes amblyopia and damages visual acuity (VA) and visual function. This study evaluated the therapeutic efficacy of perceptual learning (PL) toward improving contrast sensitivity function (CSF) and VA. A total of 25 children with LD and 25 normal children were compared in terms of CSF and VA. The LD group was further randomly allocated into two arms: nine underwent PL combined with patching and eight underwent patching only; eight patients quit the amblyopia treatment. The primary outcome was the area under log CSF (AULCSF), and the secondary outcome was the best corrected VA (BCVA). The CSF was obviously reduced in the LD group compared with that in the normal group. Moreover, the difference in the changes in the AULCSF between the PL and patching groups after 6 months of training was 0.59 (95% CI: 0.32, 0.86, p p < 0.001). Children suffering from LD with amblyopia exhibited CSF deficits and VA loss simultaneously. PL could improve CSF and VA in the amblyopic eye better than patching