Silencing transformer and transformer-2 in Zeugodacus cucurbitae causes defective sex determination with inviability of most pseudomales

transformer is a switch gene for sex determination in many insects, which cooperates with transformer-2 that is expressed in both sexes to regulate female differentiation, particularly in dipterans. Zeugodacus cucurbitae(Coquillett) is a very destructive pest worldwide, however, its sex determination pathway remains largely uncharacterized. Here, we show that the female sex ratio is sharply reduced with knockdown of either transformer or transformer-2 by RNA interference in early embryos of Z. cucurbitae. Most of the males grown from the embryos with transient transformer and transformer-2 suppression mated with wild-type females and produced mixed sex progeny, with one exception that produced only female progeny, and all of the few remaining males failed to mate with wild-type females and produced no progeny. The exceptional male and those males with mating failure were XX pseudomales as determined by the detection of Y chromosome-linked Maleness-on-the-Y, indicating that most XX pseudomales are not viable. The phenotypes of transformer and transformer-2 suggest that they play a key role in regulating sex determination and are required for female sexual development of Z. cucurbitae. Our results will be beneficial to the understanding of sex determination in Z. cucurbitae and can facilitate the development of genetic sexing strains for its biological control.

Maleness-on-the-Y(MoY) orthologue is a key regulator of male sex determination in Zeugodacus cucurbitae(Diptera:Tephritidae)

The initiation of sex differentiation in insects is regulated by primary sex determination signals.In the Medfly Ceratitis capitata and other tephritids,Maleness-on-the-Y(MoY) is the master gene for male sex determination.However,the primary signal in Zeugodacus cucurbitae(Coquillett),a very destructive tephritid pest across the world,remains ambiguous.In this study,we have isolated and characterized the Medfly MoY homolog in Z.cucurbitae,ZcMoY.ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species.ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults.Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs,giving rise to intersexes and feminization of XY individuals.The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Z.cucurbitae males.Our findings will help the understanding of sex determination in Z.cucurbitae and facilitate the development of genetic sexing strains in its biological control.

Disruption of non-classically secreted protein(MoMtp)compromised conidiation,stress homeostasis,and pathogenesis of Magnaporthe oryzae

Blast disease,caused by the hemibiotrophic ascomycete fungus,Magnaporthe oryzae,is a significant threat to sustainable rice production worldwide.Studies have shown that the blast fungus secretes vast arrays of functionally diverse proteins into the host cell for a successful disease progression.However,the final destinations of these effector proteins inside the host cell and their role in advancing fungal pathogenesis remain a mystery.Here,we reported that a putative mitochondrial targeting non-classically secreted protein(MoMtp)positively regulates conidiogenesis and appressorium maturation in M.oryzae.Moreover,MoM TP gene deletion mutant strains triggered a hypersensitive response when inoculated on rice leaves displaying that MoMtp is essential for the virulence of M.oryzae.In addition,cell wall and oxidative stress results indicated that MoMtp is likely involved in the maintenance of the structural integrity of the fungus cell.Our study also demonstrates an upregulation in the expression pattern of the MoMTP gene at all stages of infection,indicating its possible regulatory role in host invasion and the infectious development of M.oryzae.Furthermore,Agrobacterium infiltration and sheath inoculation confirmed that MoMtpGFP protein is predominantly localized in the host mitochondria of tobacco leaf and rice cells.Taken together,we conclude that MoMtp protein likely promotes the normal conidiation and pathogenesis of M.oryzae and might have a role in disturbing the proper functioning of the host mitochondria during pathogen invasion.

Antibacterial activity of bacillomycin D-like compounds isolated from Bacillus amyloliquefaciens HAB-2 against Burkholderia pseudomallei

Objective:To investigate the inhibitory effect on Burkholderia pseudomallei(B.pseudomallei)strain HNBP001 of a bacillomycin D-like cyclic lipopeptide compound named bacillomycin DC isolated from Bacillus amyloliquefaciens HAB-2.Methods:The antibacterial effect of bacillomycin DC on B.pseudomallei was determined using the disk diffusion method.The minimum inhibitory concentrations were evaluated by microdilution assay.In addition,transmission electron microscopy was performed and quantitative real-time polymerase chain reaction assay was carried out to determine the expression of Mex B,Opr D2,and qnr S genes.Results:Bacillomycin DC produced an inhibition zone against B.pseudomallei with minimum inhibitory concentration values of 12.5μg/mL 24 h after treatment and 50μg/mL at 48 and 72 h.Transmission electron microscopy showed that bacillomycin DC resulted in roughening cell surface and cell membrane damage.Quantitative real-time polymerase chain reaction analysis showed low expression of Mex B,Opr D2 and qnr S genes.Conclusions:Bacillomycin DC inhibits the growth of B.pseudomallei and can be a new candidate for antimicrobial agents of B.pseudomallei.

SUMOylation-modified Pelota-Hbs1 RNA surveillance complex restricts the infection of potyvirids in plants

RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals.However,it is not known whether two other RNA quality control pathways,nonstop decay and no-go decay,are capable of restricting viruses in plants.Here,we show that the evolutionarily conserved Pelota–Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae(termed potyvirids),the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide.Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron,which is conserved in almost all potyvirids.This allows Pelota to target the virus and act as a viral restriction factor.Furthermore,Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta.Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation.These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.

A novel ambigrammatic mycovirus,PsV5,works hand in glove with wheat stripe rust fungus to facilitate infection

Here we describe a novel narnavirus,Puccinia striiformis virus 5(PsV5),from the devastating wheat stripe rust fungus P.striiformis f.sp.tritici(Pst).The genome of PsV5 contains two predicted open reading frames(ORFs)that largely overlap on reverse strands:an RNA-dependent RNA polymerase(RdRp)and a reverseframe ORF(rORF)with unknown function.Protein translations of both ORFs were demonstrated by immune technology.Transgenic wheat lines overexpressing PsV5(RdRp-rORF),RdRp ORF,or rORF were more susceptible to Pst infection,whereas PsV5-RNA interference(RNAi)lines were more resistant.Overexpression of PsV5(RdRp-rORF),RdRp ORF,or rORF in Fusarium graminearum also boosted fungal virulence.We thus report a novel ambigrammatic mycovirus that promotes the virulence of its fungal host.The results are a significant addition to our understanding of virosphere diversity and offer insights for sustainable wheat rust disease control.

RR-1 cuticular protein TcCPR69 is required for growth and metamorphosis in Tribolium castaneum

Cuticle is not only critical for protecting insects from noxious stimuli but is also involved in a variety of metabolic activities.Cuticular proteins(CPs)affect cuticle structure and mechanical properties during insect growth,reproduction,and environmental adaptation.Here,we describe the identification and characterization of a member of the RR-1 subfamily of CPs with an R&R consensus(CPR)in Tribolium castaneum(TcCPR69).Although it was previously reported to be highly expressed in the wings,we found that knocking down TcCPR69 by RNA interference(RNAi)did not cause obvious wing abnormalities but markedly disrupted the growth and metamorphosis of beetles with 100%cumulative mortality;additionally,the chitin content of the pharate adult was decreased and the new abdominal cuticle was significantly thinner before molting.TcCPR69 showed chitin-binding ability and the expression levels of key genes involved in chitin metabolism(trehalase[TcTRE],chitin synthase[TcCHSA and TcCHSB],and chitinase[TcCHT5 and TcCHT10])were also decreased by TcCPR69 knockdown.TcCPR69 gene expression peaked shortly after molting and was increased 2.61 fold at 12 h after 20-hydroxyecdysone(20E)injection.This was reversed by RNAi of the ecdysone-related genes ecdysone receptor(TcECR)and fushi tarazu transcription factor 1(TcFTZ-F1).These results indicate that TcCPR69 is positively regulated by 20E signaling to contribute to cuticle formation and maintain chitin accumulation during the growth and metamorphosis of beetles.