RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions.

BACKGROUND: Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions. RESULTS: C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs. CONCLUSIONS: We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco.

MAIN CONCLUSION: External application of dsRNA molecules from Tobacco mosaic virus (TMV) p126 and CP genes confers significant resistance against TMV infection. Exogenously applied dsRNA exhibits a rapid systemic trafficking in planta , and it is processed successfully by DICER-like proteins producing small interfering RNAs. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA), which protects eukaryotic cells against invasive nucleic acids like viruses and transposons. In the present study, we used a non-transgenic strategy to induce RNAi in Nicotiana tabacum cv. Xanthi plants against TMV. DsRNA molecules for the p126 (TMV silencing suppressor) and coat protein (CP) genes were produced by a two-step PCR approach followed by in vitro transcription. The application of TMV p126 dsRNA onto tobacco plants induced greater resistance against TMV infection as compared to CP dsRNA (65 vs. 50 %). This study also reported the fast systemic spread of TMV p126 dsRNA from the treated (local) to non-treated (systemic) leaves beginning from 1 h post-application, confirmed by both conventional and real-time RT-PCR. Furthermore, we employed a stem-loop RT-PCR and confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves post-application. The approach employed could represent a simple and environmentally safe way for the control of plant viruses in future agriculture.

Enhanced stress resilience in potato by deletion of Parakletos.

Continued climate change impose multiple stressors on crops, including pathogens, salt, and drought, severely impacting agricultural productivity. Innovative solutions are necessary to develop resilient crops. Here, using quantitative potato proteomics, we identify Parakletos, a thylakoid protein that contributes to disease susceptibility. We show that knockout or silencing of Parakletos enhances resistance to oomycete, fungi, bacteria, salt, and drought, whereas its overexpression reduces resistance. In response to biotic stimuli, Parakletos-overexpressing plants exhibit reduced amplitude of reactive oxygen species and Ca2+ signalling, and silencing Parakletos does the opposite. Parakletos homologues have been identified in all major crops. Consecutive years of field trials demonstrate that Parakletos deletion enhances resistance to Phytophthora infestans and increases yield. These findings demark a susceptibility gene, which can be exploited to enhance crop resilience towards abiotic and biotic stresses in a low-input agriculture.

Streptomyces alleviate abiotic stress in plant by producing pteridic acids.

Soil microbiota can confer fitness advantages to plants and increase crop resilience to drought and other abiotic stressors. However, there is little evidence on the mechanisms correlating a microbial trait with plant abiotic stress tolerance. Here, we report that Streptomyces effectively alleviate drought and salinity stress by producing spiroketal polyketide pteridic acid H (1) and its isomer F (2), both of which promote root growth in Arabidopsis at a concentration of 1.3 nM under abiotic stress. Transcriptomics profiles show increased expression of multiple stress responsive genes in Arabidopsis seedlings after pteridic acids treatment. We confirm in vivo a bifunctional biosynthetic gene cluster for pteridic acids and antimicrobial elaiophylin production. We propose it is mainly disseminated by vertical transmission and is geographically distributed in various environments. This discovery reveals a perspective for understanding plant-Streptomyces interactions and provides a promising approach for utilising beneficial Streptomyces and their secondary metabolites in agriculture to mitigate the detrimental effects of climate change.

Induction of Plant Resistance in Tobacco (Nicotiana tabacum) against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco (Nicotiana tabacum) plants against TSWV. Double-stranded RNA (dsRNA) molecules for the NSs (silencing suppressor) and N (nucleoprotein) genes were produced by a two-step PCR approach followed by in vitro transcription. When topically applied to tobacco leaves, both molecules elicited a resistance response. Host response to the treatments was measured by determining the time to symptom expression, and the level of resistance by absolute quantification of the virus. We also show the systemic movement of dsRNA_N from the inoculated leaves to younger, non-inoculated leaves. Post-application, viral siRNAs were detected for up to nine days in inoculated leaves and up to six days in non-inoculated leaves. The topical application of dsRNAs to induce RNAi represents an environmentally safe and efficient way to manage TSWV in tobacco crops and could be applicable to other TSWV-susceptible crops.

dsRNA Molecules From the Tobacco Mosaic Virus p126 Gene Counteract TMV-Induced Proteome Changes at an Early Stage of Infection.

Exogenous application of double-stranded RNA (dsRNA) in the tobacco-Tobacco mosaic virus (TMV) pathosystem was shown previously to induce resistance against TMV providing an alternative approach to transgenesis. In the present study, we employed proteomics technology to elucidate the effect of TMV on tobacco as well as the effect of exogenous application of TMV p126 dsRNA molecules (dsRNAp126) at an early stage of the tobacco-TMV interaction. The proteome of tobacco leaf at 15 min post inoculation (mpi) in the presence or absence of dsRNAp126 molecules was studied. Thirty-six tobacco proteins were differentially accumulated in TMV-infected vs. healthy tobacco leaf tissue. The identified main differential TMV-responsive proteins were found to be involved in photosynthesis, energy metabolism, stress, and defense responses. Most of the virus-induced changes in the tobacco leaf proteome were not observed in the leaves treated with dsRNAp126 + TMV. The results indicated that the protein changes induced by TMV infection were counteracted by the exogenous application of dsRNAp126 molecules. Moreover, using small RNA sequencing, we showed that the exogenously applied dsRNAp126 was efficiently processed in tobacco as early as 15 min post application (mpa) to produce small interfering RNAs (siRNAs); the dicing pattern was not affected by the presence of TMV. The presence of dsRNAp126 reduced TMV p126 RNA abundance suggesting virus titer reduction via a sequence-specific mechanism, since a non-homologous dsRNA did not protect from TMV infection nor affect TMV accumulation.

High Throughput Sequencing-Aided Survey Reveals Widespread Mixed Infections of Whitefly-Transmitted Viruses in Cucurbits in Georgia, USA.

Viruses transmitted by the sweet potato whitefly (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected and small RNA libraries were prepared and sequenced from three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV, were assembled from the small RNA sequences. An analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region.