Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV–planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes. Numerous parasites including viruses, bacteria, and microsporidia can be maternally transmitted, with the parasite passing from mother to offspring, usually through eggs. However, the process of the parasites spreading into eggs from primarily infected tissues and the factors that mediate this process in live hosts or vectors are unknown due to the lack of useful tools. Here, we used several techniques to investigate the molecular mechanisms of transovarial transmission of Rice stripe virus (RSV), a plant virus belonging to the genus Tenuivirus, by its insect vector (Laodelphax striatellus). We found that the nucleocapsid protein of RSV bound to insect's vitellogenin (Vg) in vitro and in vivo. We also found that RSV invaded the egg tubes of the ovariole until Vg is highly expressed, then colocalized with Vg in the germarium. When Vg expression was knocked down due to RNA interference, the invasion of ovarioles by RSV decreased largely. Our study provides new insights into the transovarial transmission of an important viral pathogen that uses existing transovarial transportation systems in insect vectors to invade eggs.

Plant RNA-dependent RNA polymerase 1(RDR1) is a key component of antiviral RNA silencing pathway contributing to the biogenesis of the virus-derived small interfering RNAs (vsiRNAs). This enzyme is also responsible for producing virus-activated endogenous siRNAs (vasiRNAs) to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific miRNA, miR444, is a key factor to relay the antiviral signaling from virus infection to OsRDR1 expression. Expression of miR444 is enhanced by infection of Rice stripe virus (RSV) and overexpression of miR444 improves rice resistance against RSV infection accompanied by upregulation of OsRDR1 expression. We further show that three miR444 targets, the MIKCC-type MADS-box proteins OsMADS23, OsMADS27a and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a and OsMADS57 on OsRDR1 transcription thus to activate OsRDR1-dependent antiviral RNA silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results unveil a molecular cascade in the rice antiviral pathway in which miR444 and its MADS-box targets directly control OsRDR1 transcription.

As one of the largest gene families, F-box domain proteins have important roles in regulating various developmental processes and stress responses. In this study, we have investigated a rice F-box domain gene, MAIF1. The MAIF1 protein is mainly localized in the plasma membrane and nucleus. MAIF1 expression is induced rapidly and strongly by abscisic acid (ABA) and abiotic stresses. MAIF1 expression is also induced in root tips by sucrose, independent of its hydrolytic hexose products, glucose and fructose, and the plant hormones auxin and cytokinin. Overexpression of MAIF1 reduces rice ABA sensitivity and abiotic stress tolerance and promotes rice root growth. These results suggest that MAIF1 is involved in multiple signaling pathways in regulating root growth. Growth restraint in plants is an acclimatization strategy against abiotic stress. Our results also suggest that MAIF1 plays the negative role in response to abiotic stress possibly by regulating root growth.

Summary: Cucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b‐mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA‐directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24‐nt repeat‐associated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24‐nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4‐related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4‐related functions. We propose that by adopting novel counter‐host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.

Huanglongbing (HLB) is a devastating citrus disease. In this work, we report an HLB resistance regulatory circuit in Citrus composed of an E3 ubiquitin ligase, PUB21, and its substrate, the MYC2 transcription factor, which regulates jasmonate-mediated defense responses. A helitron insertion in the PUB21 promoter introduced multiple MYC2-binding cis-elements to create a regulatory circuit linking the PUB21 activity with MYC2 degradation. Ectopic expression of a natural dominant-negative PUB21 paralog discovered in distant Citrus relatives stabilized MYC2 and conferred resistance to HLB. Antiproteolysis peptides (APPs), identified by artificial intelligence, stabilized MYC2 by binding and inhibiting PUB21 activity. A 14–amino acid peptide, APP3-14, molecularly controlled HLB in greenhouse and field trials. This approach represents a strategy to combat uncultivable pathogens through targeted disease resistance protein stabilization.