A receptor-like kinase recognizes viral proteins at the trans-Golgi network/early endosome and inhibits infection in rice

Plant cells are equipped with a collection of membrane surface molecular “antennas” specifically sensitive to different signals. They are mostly represented by hundreds of receptor-like kinases (RKs): about 600 encoded in the Arabidopsis genome (1), which allow plants to react swiftly to signals related to the progress of their own ontogeny (intercellular communication) and also to environmental changes, including stress situations or pathogen attack. Such surface alertness is especially important for sessile organisms bound to be born and die at the same single spot. Not surprisingly, study of plant RK regulation is among the most important current fields of plant research. RKs involved in pathogen presence recognition via specific binding of pathogen activity-related molecular species—pattern-recognition receptor kinases (PRKs)—are also studied for practical reasons of plant protection (for a recent overview, see refs. 2⇓–4). RKs, as most other components of the plasmalemma (PM), are not static. Even without activation, RKs undergo constitutive recycling to and from the PM by insertion (exocytosis) and removal (endocytosis), often involving the trans-Golgi network/early endosome (TGN/EE). Kinetics and steady-state localization differs for individual RK species (5, 6). Until recently, there was only very limited insight into what happens to PRKs in plant cells upon the arrival of the signal: that is, after specific ligand binding resulting in an intracellular kinase domain activation and signaling initiation. Two side-by-side reports in PNAS (7, 8) show that three different PRKs [PEP receptor 1 (PEPR1), EF-TU RECEPTOR (EFR), and FLAGELLIN SENSING 2 (FLS2)], involved in biotic defense interactions activated by three different ligands, are all removed from the cell surface via clathrin-mediated endocytosis (CME). An interacting coreceptor, BRI1-ASSOCIATED KINASE 1 (BAK1), is necessary for the internalization of all these activated receptors. Requirement for the active RK domain was demonstrated for the FLS2 PRK, but this …

CRK28, a cysteine-rich receptor-like kinase, plays a role in root organogenesis and overall growth of plants and antagonizes abscisic acid response in seed germination and primary root growth. Receptor-like kinases (RLK) orchestrate development and adaptation to environmental changes in plants. One of the largest RLK groups comprises cysteine-rich receptor-like kinases (CRKs), for which the function of most members remains unknown. In this report, we show that the loss of function of CRK28 led to the formation of roots that are longer and more branched than the parental (Col-0) plantlets, and this correlates with an enhanced domain of the mitotic reporter CycB1:uidA in primary root meristems, whereas CRK28 overexpressing lines had the opposite phenotype, including slow root growth and reduced lateral root formation. Epidermal cell analyses revealed that crk28 mutants had reduced root hair length and increased trichome number, whereas 35S::CRK28 lines present primary roots with longer root hairs but lesser trichomes in leaves. The overall growth in soil of crk28 mutant and CRK28 overexpressing lines was reduced or enhanced, respectively, when compared to the parental (Col-0) seedlings, while germination, root growth and expression analyses of ABI3 and ABI5 further showed that CRK28 modulates ABA responses, which may be important to fine-tune plant morphogenesis. Our study unravels the participation of RLK signaling in root growth and epidermal cell differentiation.

The amount of Rice stripe virus (RSV) maintained through transovarial transmission was analyzed during the development and reproduction of its vector, Laodelphax striatellus. Reverse transcription quantitative PCR analysis was used to quantify RNA expressed from the RSV coat protein (CP) gene as an estimate of RSV content in nymphs and adults of L. striatellus at various developmental stages. The 18S ribosome RNA gene of L. striatellus was chosen as the reference for calculating RSV CP expression using the comparative Ct method. Based on the CP transcript levels, the amount of RSV did not differ significantly throughout the nymphal stage or between adult females of different ages; however, RSV content tended to increase slightly as males became older. The average RSV content in males was 1.30-2.49 times that in females. The amount of RSV in L. striatellus adults was compared between generations. The RSV content of female adults did not differ significantly between the parent and progeny populations three of three different females. L. striatellus grown to adults on a susceptible cultivar and five RSV-resistant cultivars were compared to analyze whether the amount of RSV varied among cultivars. Although the amount of RSV in L. striatellus adults differed significantly among the six rice cultivars evaluated, the difference seemed independent of whether resistance genes were present. In addition, the percentage of viruliferous insects was similar among cultivars.

Planar morphogenesis, a distinct feature of multicellular organisms, is crucial for the development of ovule, progenitor of seeds. Both receptor-like kinases (RLKs) such as STRUBBELIG (SUB) and auxin gradient mediated by PIN-FORMED1 (PIN1) play instructive roles in this process. Fine-tuned intercellular communications between different cell layers during ovule development demands dynamic membrane distribution of these cell-surface proteins, presumably through vesicle-mediated sorting. However, the way it’s achieved and the trafficking routes involved are obscure. We report that HAPLESS13 (HAP13)-mediated trafficking of SUB is critical for ovule development. HAP13 encodes the μ subunit of adaptor protein 1 (AP1) that mediates protein sorting at the trans-Golgi network/early endosome (TGN/EE). The HAP13 mutant, hap13-1, is defective in outer integument growth, resulting in exposed nucellus accompanied with impaired pollen tube guidance and reception. SUB is mis-targeted in hap13-1. However, unlike that of PIN2, the distribution of PIN1 is independent of HAP13. Genetic interference of exocytic trafficking at the TGN/EE by specifically downregulating HAP13 phenocopied the defects of hap13-1 in SUB targeting and ovule development, supporting a key role of sporophytically expressed SUB in instructing female gametogenesis.

Interaction between Rice stripe virus Disease-Specific Protein and Host PsbP Enhances Virus Symptoms

Viral infection commonly induces autophagy, leading to antiviral responses or conversely, promoting viral infection or replication. In this study, using the experimental plant Nicotiana benthamiana, we demonstrated that the rice stripe virus (RSV) coat protein (CP) enhanced autophagic activity through interaction with cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 (GAPC2), a negative regulator of plant autophagy that binds to an autophagy key factor, autophagy-related protein 3 (ATG3). Competitive pull-down and co-immunoprecipitation (Co-IP)assays showed that RSV CP activated autophagy by disrupting the interaction between GAPC2 and ATG3. An RSV CP mutant that was unable to bind GAPC2 failed to disrupt the interaction between GAPC2 and ATG3 and therefore lost its ability to induce autophagy. RSV CP enhanced the autophagic degradation of a viral movement protein (MP) encoded by a heterologous virus, citrus leaf blotch virus (CLBV). However, the autophagic degradation of RSV-encoded MP and RNA-silencing suppressor (NS3) proteins was inhibited in the presence of CP, suggesting that RSV CP can protect MP and NS3 against autophagic degradation. Moreover, in the presence of MP, RSV CP could induce the autophagic degradation of a remorin protein (NbREM1), which negatively regulates RSV infection through the inhibition of viral cell-to-cell movement. Overall, our results suggest that RSV CP induces a selective autophagy to suppress the antiviral factors while protecting RSV-encoded viral proteins against autophagic degradation through an as-yet-unknown mechanism. This study showed that RSV CP plays dual roles in the autophagy-related interaction between plants and viruses.

Rice stripe virus (RSV) causes rice stripe disease, which is one of the most serious rice diseases in eastern Asian countries. It has been shown that overexpression of RSV coat protein (CP) in rice plants enhances resistance against virus infection. However, the detailed mechanism underlying RSV CP-mediated virus resistance remains to be determined. In this study, we show that both translatable and non-translatable RSV CP transgenic Arabidopsis plants exhibited immunity to virus infection. By using deep sequencing analysis, transgene-derived small interfering RNAs (t-siRNAs) from non-translatable CP transgenic plants and virus-derived small interfering RNAs (vsiRNAs) mapping in the CP region from RSV-infected wild-type plants showed similar sequence distribution patterns, except for a significant increase in the abundance of t-siRNA reads compared with that of CP-derived vsiRNAs. To further test the correlation of t-siRNAs with RSV immunity, we developed RSV CP transgenic Arabidopsis plants in an siRNA-deficient dcl2/3/4 mutant background, and these CP transgenic plants showed the same sensitivity to RSV infection as non-transgenic plants. Together, our data indicate that the expression of RSV CP protein from a transgene is not a prerequisite for virus resistance and RSV CP-mediated resistance is mostly associated with the RNA silencing mechanism in Arabidopsis plants.

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

The jasmonic acid (JA) pathway plays crucial roles in plant defence against pathogens and herbivores. Rice stripe virus (RSV) is the type member of the genus Tenuivirus. It is transmitted by the small brown planthopper (SBPH) and causes damaging epidemics in East Asia. The role(s) that JA may play in the tripartite interaction against RSV, its host, and vector are poorly understood. Here, we found that the JA pathway was induced by RSV infection and played a defence role against RSV. The coat protein (CP) was the major viral component responsible for inducing the JA pathway. Methyl jasmonate treatment attracted SBPHs to feed on rice plants while a JA‐deficient mutant was less attractive than wild‐type rice. SBPHs showed an obvious preference for feeding on transgenic rice lines expressing RSV CP. Our results demonstrate that CP is an inducer of the JA pathway that activates plant defence against RSV while also attracting SBPHs to feed and benefitting viral transmission. This is the first report of the function of JA in the tripartite interaction between RSV, its host, and its vector.

Nonstructural protein 3 (NS3) encoded by RNA3 of Rice stripe virus (RSV), known to be a suppressor of gene silencing, was cloned and sequenced. The cloned NS3 gene is composed of 636 nucleotides encoding 211 deduced amino acids, and showed a high degree of similarity with the equivalent genes isolated from Korea, Japan and China. The NS3 gene promoted the enhancement of transient gene expression and suppressed transgene co-silencing. In the transient GFP expression via agroinfiltration, GFP expression was dramatically enhanced in terms of both protein yield and expression period in the presence of NS3. The highest accumulation of GFP protein reached to 6.8% of total soluble proteins, which corresponded to a two-fold increase compared to that obtained in the absence of NS3. In addition, NS3 significantly suppressed the initiation of GFP co-silencing induced by the additive GFP infiltration in GFP-transgenic Nicotiana benthamiana. The NS3 gene was also found to be a stronger suppressor than Cucumber mosaic virus 2b. These observations are believed to be derived from the strong suppressive effect of NS3 on gene silencing, and indicate that NS3 could be used as an effective enhancer for the rapid production of foreign proteins in plants.

Viruses are encapsidated mobile genetic elements that rely on host cells for replication. Several cytoplasmic RNA viruses synthesize proteins and/or RNAs that translocate to infected cell nuclei. However, the underlying mechanisms and role(s) of cytoplasmic-nuclear trafficking are unclear. We demonstrate that infection of small brown planthoppers with rice stripe virus (RSV), a negarnaviricot RNA virus, results in K63-linked polyubiquitylation of RSV's nonstructural protein 3 (NS3) at residue K127 by the RING ubiquitin ligase (E3) LsRING. In turn, ubiquitylation leads to NS3 trafficking from the cytoplasm to the nucleus, where NS3 regulates primary miRNA pri-miR-92 processing through manipulation of the microprocessor complex, resulting in accumulation of upregulated miRNA lst-miR-92. We show that lst-miR-92 regulates the expression of fibrillin 2, an extracellular matrix protein, thereby increasing RSV loads. Our results highlight the manipulation of intranuclear, cytoplasmic, and extracellular components by an RNA virus to promote its own replication in an insect vector.

Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 (K372E) with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network.

MicroRNAs (miRNAs) are small regulatory RNAs processed from primary miRNA transcripts, and plant miRNAs play important roles in plant growth, development, and response to infection by microbes. Microbial infections broadly alter miRNA biogenesis, but the underlying mechanisms remain poorly understood. In this study, we report that the Rice stripe virus (RSV)-encoded nonstructural protein 3 (NS3) interacts with OsDRB1, an indispensable component of the rice (Oryza sativa) miRNA-processing complex. Moreover, the NS3-OsDRB1 interaction occurs at the sites required for OsDRB1 self-interaction, which is essential for miRNA biogenesis. Further analysis revealed that NS3 acts as a scaffold between OsDRB1 and pri-miRNAs to regulate their association and aids in vivo processing of pri-miRNAs. Genetic evidence in Arabidopsis showed that NS3 can partially substitute for the function of double-stranded RNA binding domain (dsRBD) of AtDRB1/AtHYL1 during miRNA biogenesis. As a result, NS3 induces the accumulation of several miRNAs, most of which target pivotal genes associated with development or pathogen resistance. In contrast, a mutant version of NS3 (mNS3), which still associated with OsDRB1 but has defects in pri-miRNA binding, reduces accumulation of these miRNAs. Transgenic rice lines expressing NS3 exhibited significantly higher susceptibility to RSV infection compared with non-transgenic wild-type plants, whereas the transgenic lines expressing mNS3 showed a less-sensitive response. Our findings revealed a previously unknown mechanism in which a viral protein hijacks OsDRB1, a key component of the processing complex, for miRNA biogenesis and enhances viral infection and pathogenesis in rice.

Rice stripe virus (RSV) is the causative agent of rice stripe disease and is completely dependent on insect vectors for its plant-to-plant transmission. Laodelphax striatellus is the major insect vector for RSV. In this study, we explored the interactions between RSV infection and L. striatellus autophagy, a potential intrinsic antiviral mechanism in insects. We found that L. striatellus autophagic activity did not affect RSV infection; however, the autophagy-related-8 (Atg8) gene significantly enhanced virus infection. During RSV initial infection within the L. striatellus midgut, silencing of Atg8 expression significantly decreased the phosphorylation of c-Jun N-terminal kinase (p-JNK); however, when RSV infection is absent, silencing of Atg8 did not alter p-JNK levels. These results indicated that Atg8 might activate the JNK machinery by allowing more virus infection into cells. We further revealed that Atg8-deficiency significantly decreased RSV accumulation on the surface of the insect midgut epithelial cells, suggesting a receptor trafficking function of the γ-aminobutyric acid receptor-associated protein family. Using the RSV ovary entry as a model, in which vitellogenin receptor (VgR) mediates RSV cell entry, we clarified that Atg8-deficiency decreased the abundance of VgR localizing on the cytomembrane and disturbed the attachment of RSV in the germarium zones. Collectively, these results revealed an autophagy-independent function of L. striatellus Atg8 that enhances RSV initial infection by increasing virus attachment on the infection sites.

Understanding the role of cysteine-rich receptor-like kinases (CRKs) in plant defense mechanisms is crucial for enhancing wheat resistance to leaf rust fungus infection. Here, we identified and verified 164 members of the CRK gene family using the Triticum aestivum reference version 2 collected from the international wheat genome sequencing consortium (IWGSC). The proteins exhibited characteristic features of CRKs, including the presence of signal peptides, cysteine-rich/stress antifungal/DUF26 domains, transmembrane domains, and Pkinase domains. Phylogenetic analysis revealed extensive diversification within the wheat CRK gene family, indicating the development of distinct specific functional roles to wheat plants. When studying the expression of the CRK gene family in near-isogenic lines (NILs) carrying Lr57- and Lr14a-resistant genes, Puccinia triticina, the causal agent of leaf rust fungus, triggered temporal gene expression dynamics. The upregulation of specific CRK genes in the resistant interaction indicated their potential role in enhancing wheat resistance to leaf rust, while contrasting gene expression patterns in the susceptible interaction highlighted potential susceptibility associated CRK genes. The study uncovered certain CRK genes that exhibited expression upregulation upon leaf rust infection and the Lr14a-resistant gene. The findings suggest that targeting CRKs may present a promising strategy for improving wheat resistance to rust diseases.