Small RNA deep sequencing reveals the presence of multiple viral infections in cucurbit crops in Guangdong,China

Viral diseases are among the most critical damaging factors that impose a global threat to the cucurbit industry.China is the world’s leading country for the production and consumption of cucurbits.Guangdong,a province in southern China dominated by the tropical and subtropical climate,favors the survival of different plant viruses and their vectors.Five main cucurbit crops showing various disease symptoms were surveyed and collected to identify viruses infecting cucurbits in Guangdong during 2018–2020.In the field,the incidence ranged from 5-30%,or even 60-100% in the case of severely infected cucurbits.A total of 357 symptomatic samples were collected and subsequently screened for cucurbit viruses by small RNA deep sequencing and assembly(sRSA).Seventeen virus species belonging to 10 genera were identified in the five main cucurbit crops.The most common viruses were papaya ringspot virus(PRSV;Potyvirus),zucchini tigre mosaic virus(ZTMV;Potyvirus),zucchini yellow mosaic virus(ZYMV;Potyvirus),and watermelon silver mottle virus(WSMoV;Orthotospovirus),with infection rates of 24.4,19.0,17.1,and 14.3%,respectively.Notably,the most prevalent viruses were melon yellow spot orthotospovirus(MYSV)in cucumber,PRSV in squash,cucumber green mottle mosaic virus(CGMMV;Tobamovirus)in bottle gourd,WSMoV in white gourd,and ZYMV in luffa.Mixed infections were prevalent,and the types of mixed infections varied substantially in different cucurbit crops.Moreover,the full-length nucleotide sequences of watermelon green mottle mosaic virus(WGMMV),CGMMV,and watermelon virus A(WVA;Wamavirus)identified in bottle gourd were cloned and analyzed.This study is the first reporting WGMMV infecting bottle gourd in China mainland.In summary,the results demonstrate that in Guangdong,the most prevalent viruses belong to potyviruses,orthotospoviruses,and tobamoviruses groups.The findings will facilitate agricultural researchers and farmers to plan and implement effective disease control strategies aiming at timely detection and management of cucurbit-infecting viral pathogens.

Identification of novel salt stress-responsive microRNAs through sequencing and bioinformatic analysis in a unique halophilic Dunaliella salina strain

Dunaliella salina is a classic halophilic alga.However,its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown.A unique halophilic alga strain,DS-CN1,was screened from four D.salina strains via cell biological,physiological,and biochemical methods.High-throughput sequencing of small RNAs(sRNAs)of DS-CN1 in culture medium containing 3.42-mol/L NaCl(SS group)or 0.05-mol/L NaCl(CO group)was performed on the BGISEQ-500 platform.The annotation and sequences of D.salina sRNAs were profiled.Altogether,44 novel salt stress-responsive microRNAs(miRNAs)with a relatively high C content,with the majority of them being 24 nt in length,were identified and characterized in DS-CN1.Twenty-one differentially expressed miRNAs(DEMs)in SS and CO were screened via bioinformatic analysis.A total of 319 putative salt stress-related genes targeted(104 overlapping genes)by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research.Furthermore,these target genes were classified and enriched by GO and KEGG pathway analysis.Moreover,5 novel DEMs(dsa-mir3,dsa-mir16,dsa-mir17,and dsa-mir26 were significantly upregulated,and dsa-mir40 was significantly downregulated)and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR.Next,their regulatory relationships were comprehensively analyzed.Lastly,a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs.Taken together,our results suggest that 44 novel salt stress-responsive microRNAs were identified,and 4 of them might play important roles in D.salina upon salinity stress and contribute to clarify its distinctive halophilic feature.Our study will shed light on the regulatory mechanisms of salt stress responses.

Small RNA deep sequencing reveals full-length genome of Citrus yellow vein clearing virus in Chongqing,China

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-de- rived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study. Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus （CYVCV） isolate YN were obtained using the small RNA deep sequencing technology. Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 （JX040635） and YN （KP313242）, both of which belong to the genus Mandarivirus in the family Alphaflexiviridae. Mapping analysis of viral-derived siRNA （vsiRNA） profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority. BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon. This is the first CYVCV isolate detected in Chongqing and the second in China. This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region.

Global Annotation of Small RNA and MicroRNA Mature Sequences from Developing Ovules of Gossypium hirsutum L.

The involvement of small RNAs in cotton fiber development is under explored.The objective of this work was to directly clone,annotate,and analyze small RNAs of developing ovules to reveal

HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells

BACKGROUND The role of exosomes derived from HepG2.2.15 cells,which express hepatitis B virus(HBV)-related proteins,in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive.The focus was on comprehending the relationship and influence of differentially expressed microRNAs(DE-miRNAs)within these exosomes.AIM To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell(HSC)LX2 and the progression of liver fibrosis.METHODS Exosomes from HepG2.2.15 cells,which express HBV-related proteins,were isolated from parental HepG2 and WRL68 cells.Western blotting was used to confirm the presence of the exosomal marker protein CD9.The activation of HSCs was assessed using oil red staining,whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells.Additionally,we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2′-deoxyuracil staining and western blotting,respectively.DE-miRNAs were analyzed using DESeq2.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were used to annotate the target genes of DE-miRNAs.RESULTS Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells.A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells.GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation,intracellular signal transduction,negative regulation of apoptosis,extracellular exosomes,and RNA binding.KEGG pathway analysis highlighted ubiquitin-mediated proteolysis,the MAPK signaling pathway,viral carcinogenesis,and the toll-like receptor signaling pathway,among others,as enriched in these targets.CONCLUSION These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation,proliferation,and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Identification and Characterization of Drought Stress-Responsive Novel microRNAs in Dongxiang Wild Rice

MicroRNAs （miRNAs） are non-coding small RNAs, which play important regulatory roles in response to biotic and abiotic stresses. Dongxiang wild rice （Oryza rufipogon, DXWR） can survive in extreme drought environment, but its molecular mechanism of drought resistance is still largely unknown. To further explore miRNA regulatory mechanisms involved in drought resistance, we identified 138 novel miRNAs in DXWR using small RNA sequencing and bioinformatics approaches, and found that the expression levels of 67 novel miRNAs were significantly affected by drought stress. In total, 200 candidate target genes were predicted and annotated for the drought stress-responsive novel miRNAs. Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways suggested that most of the target genes were related to metabolism. Stem-loop quantitative real-time PCR （qRT-PCR） results exhibited high concordance with sequencing data, which confirmed that miRNA expression patterns based on small RNA sequencing in the present study were reliable. Meanwhile, qRT-PCR validated the inverse expression patterns between several miRNAs and their target genes. These results will enhance our understanding of miRNA regulatory mechanisms in response to drought stress in DXWR, and can serve as an important reference for the protection and utilization of this valuable genetic resource.

Liver injury changes the biological characters of serum small extracellular vesicles and reprograms hepatic macrophages in mice

BACKGROUND Serum small extracellular vesicles(sEVs)and their small RNA(sRNA)cargoes could be promising biomarkers for the diagnosis of liver injury.However,the dynamic changes in serum sEVs and their sRNA components during liver injury have not been well characterized.Given that hepatic macrophages can quickly clear intravenously injected sEVs,the effect of liver injury-related serum sEVs on hepatic macrophages deserves to be explored.AIM To identify the characteristics of serum sEVs and the sRNAs during liver injury and explore their effects on hepatic macrophages.METHODS To identify serum sEV biomarkers for liver injury,we established a CCL4-induced mouse liver injury model in C57BL/6 mice to simulate acute liver injury(ALI),chronic liver injury(CLI)and recovery.Serum sEVs were obtained and characterized by transmission electron microscopy and nanoparticle tracking analysis.Serum sEV sRNAs were profiled by sRNA sequencing.Differentially expressed microRNAs(miRNAs)were compared to mouse liver-enriched miRNAs and previously reported circulating miRNAs related to human liver diseases.The biological significance was evaluated by Ingenuity Pathway Analysis of altered sEV miRNAs and conditioned cultures of ALI serum sEVs with primary hepatic macrophages.RESULTS We found that both ALI and CLI changed the concentration and morphology of serum sEVs.The proportion of serum sEV miRNAs increased upon liver injury,with the liver as the primary contributor.The altered serum sEV miRNAs based on mouse studies were consistent with human liver disease-related circulating miRNAs.We established serum sEV miRNA signatures for ALI and CLI and a panel of miRNAs(miR-122-5p,miR-192-5p,and miR-22-3p)as a common marker for liver injury.The differential serum sEV miRNAs in ALI contributed mainly to liver steatosis and inflammation,while those in CLI contributed primarily to hepatocellular carcinoma and hyperplasia.ALI serum sEVs decreased both CD86 and CD206 expression in monocyte-derived macrophages but increased CD206 expression in resident macrophages in vitro.CONCLUSION Serum sEVs acquired different concentrations,sizes,morphologies and sRNA contents upon liver injury and could change the phenotype of liver macrophages.Serum sEVs therefore have good diagnostic and therapeutic potential for liver injury.

Investigation on Viral Pathogens in <i>Vitis vinifera</i>from Four Production Bases in Hangzhou Vicinity of China by sRNAseq and Molecular Validation

This is the first systematic investigation of viral pathogens in Vitis vinifera from Hangzhou vicinity of China. About 7 viruses and 5 viroids were annotated from four production bases “Dushicun”, “Wangjiayuan”, “Xiajiangcun”, and “Yangducun” covering 15 cultivars through sRNAseq technique. At least 3 viruses—grapevine leaf roll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV) and grapevine geminivirus A (GGVA), and 4 viroids—hop stunt viroid (HSVd), citrus viroid II (CVd-II), grapevine yellow speckle viroid 1 (GYSVd-1) and grapevine yellow speckle viroid 2 (GYSVd-2) infected all four bases. “Yangducun” base showed 11, the most infected pathogens. GYSVd-1 showed the highest accumulation in host of Wangjiayuan base. The main infected pathogens were verified by reverse-transcription polymerase chain reaction (RT-PCR) technique, the detected rate reached to 85% - 100%. The results provide an important basis for effective and precise detection of viral diseases in the area and for the virus-free cultivation in future.

A 5′tRNA-Ala-derived small RNA regulates anti-fungal defense in plants

Apart from their primordial role in protein synthesis,t RNAs can be cleaved to produce t RNA-derived small RNAs(ts RNAs).The biological functions of ts RNAs in plants remain largely unknown.In this study,we developed Rtc B ligation-based small RNA(s RNA)sequencing,a method that captures and distinguishes between 3′-2′,3′-cyclic-phosphate(c P)/phosphate(P)-terminated s RNAs and 3′-OH-terminated s RNAs,and profiled 5′ts RNAs and 5′t RNA halves in Arabidopsis thaliana.We found that Arabidopsis 5′ts RNAs and 5′t RNA halves predominantly contain a c P at the 3′end and require S-like RNase 1(RNS1)and RNS3 for their production.One of the most abundant 5′ts RNAs,5′ts R-Ala,by associating with AGO1,negatively regulates Cytochrome P45071 A13(CYP71 A13)expression and camalexin biosynthesis to repress anti-fungal defense.Interestingly,5′ts R-Ala is downregulated upon fungal infection.Our study provides a global view of 5′ts RNAs and 5′t RNA halves in Arabidopsis and unravels an important role of a 5′ts RNA in regulating anti-fungal defense.