Zongtao Sun

Genome-Wide Characterization of Rice Black Streaked Dwarf Virus-Responsive MicroRNAs in Rice Leaves and Roots by Small RNA and Degradome Sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs which typically function by guiding cleavage of target mRNAs. They play important roles in development, abiotic stress and responses to pathogens. Four small RNA libraries and four degradome libraries were constructed from the leaves and roots of healthy rice and plants infected with Rice black streaked dwarf virus (RBSDV). Analysis of the deep sequencing results showed that the expression patterns of 14 miRNAs in leaves and 16 miRNAs in roots changed significantly in response to RBSDV infection. Some responses were similar in roots and leaves, but many miRNAs responded differently in different tissues. The results were confirmed for selected miRNAs by quantitative real-time PCR. By using degradome sequencing, a total of 104 target transcripts for 17 conserved and 16 non-conserved miRNAs were shown to be responsive to RBSDV infection. Fifteen novel miRNAs were also identified by small RNA and degradome sequencing. The results provide new insights into the regulatory networks of miRNAs and their targets in different plant tissues in response to virus infection.

Rice black-streaked dwarf virus P10 induces membranous structures at the ER and elicits the unfolded protein response in Nicotiana benthamiana.

Endoplasmic reticular (ER) membrane modifications play an important role in viral RNA replication and virion assembly but little is known about the involvement of ER-membrane remodeling in the infection cycle of fijiviruses in plant cells. The subcellular localization of Rice black-streaked dwarf virus outer capsid P10 was therefore examined using live-cell imaging. P10 fused to eGFP formed vesicular structures associated with ER membranes in Nicotiana benthamiana epidermal cells and in rice protoplasts. Subcellular fractionation experiments confirmed that P10 is an integral membrane protein. Three predicted transmembrane domains and two less-well-defined domains were each able to target eGFP to the ER. Disruption of the actin cytoskeleton with LatB, indicated that the maintenance of P10-induced membrane structures required the intact actin cytoskeleton. P10 induced the expression of ER stress marker genes, including ER stress-related chaperones and transcription factor, indicating that RBSDV P10 triggers ER stress and the unfolded protein response.

The secretory pathway and the actomyosin motility system are required for plasmodesmatal localization of the P7-1 of rice black-streaked dwarf virus

Rice black-streaked dwarf virus (RBSDV), a plant-infecting reovirus (genus Fijivirus), generally induces virus-containing tubules in infected cells. The nonstructural protein P7-1, encoded by the first open reading frame of segment 7, is involved in forming the structural matrix of these tubules. In experiments to investigate the subcellular localization of P7-1 in Nicotiana benthamiana epidermal cells, fluorescence of P7-1:eGFP was observed in the nucleus, cytoplasm and cell periphery, and in punctate points along the cell wall of plasmolyzed cells. Co-localization with plasmodesmata-located protein 1 showed that P7-1 formed the punctate points at plasmodesmata. Mutational analysis demonstrated that transmembrane domain 1 and adjacent residues were necessary and sufficient for P7-1 to form punctate structures at the cell wall in the plasmolyzed cells. Chemical drug and protein inhibitor treatments indicated that P7-1 utilized the ER-to-Golgi secretory pathway and the actomyosin motility system for its intracellular transport. The plasmodesmatal localization of RBSDV P7-1 is therefore dependent on the secretory pathway and the actomyosin motility system.

Identification and profiling of conserved and novel microRNAs in Laodelphax striatellus in response to rice black-streaked dwarf virus (RBSDV) infection.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in various biological processes. This study examined microRNA profiles of Laodelphax striatellus using the small RNA libraries derived from virus free (VF) and rice black-streaked dwarf virus (RBSDV) infected (RB) insects. A total of 59 mature miRNAs (46 miRNA families) were identified as conserved insect miRNAs in both VF and RB libraries. Among these conserved miRNAs, 24 were derived from the two arms of 12 miRNA precursors. Nine conserved L. striatellus miRNAs were up-regulated and 12 were down-regulated in response to RBSDV infection. In addition, a total of 20 potential novel miRNA candidates were predicted in the VF and RB libraries. The miRNA transcriptome profiles and the identification of L. striatellus miRNAs differentially expressed in response to RBSDV infection will contribute to future studies to elucidate the complex miRNA-mediated regulatory network activated by pathogen challenge in insect vectors.

Jasmonic acid-mediated defense suppresses brassinosteroid-mediated susceptibility to Rice black streaked dwarf virus infection in rice

Plant hormones play a vital role in plant immune responses. However, in contrast to the relative wealth of information on hormone-mediated immunity in dicot plants, little information is available on monocot-virus defense systems. We used a high-throughput-sequencing approach to compare the global gene expression of Rice black-streaked dwarf virus (RBSDV)-infected rice plants with that of healthy plants. Exogenous hormone applications and transgenic rice were used to test RBSDV infectivity and pathogenicity. Our results revealed that the jasmonic acid (JA) pathway was induced while the brassinosteroid (BR) pathway was suppressed in infected plants. Foliar application of methyl jasmonate (MeJA) or brassinazole (BRZ) resulted in a significant reduction in RBSDV incidence, while epibrassinolide (BL) treatment increased RBSDV infection. Infection studies using coi1-13 and Go mutants demonstrated JA-mediated resistance and BR-mediated susceptibility to RBSDV infection. A mixture of MeJA and BL treatment resulted in a significant reduction in RBSDV infection compared with a single BL treatment. MeJA application efficiently suppressed the expression of BR pathway genes, and this inhibition depended on the JA coreceptor OsCOI1. Collectively, our results reveal that JA-mediated defense can suppress the BR-mediated susceptibility to RBSDV infection.

ARGONAUTE SUBFAMILY GENES IN THE SMALL BROWN PLANTHOPPER, Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Argonaute (AGO) proteins are essential catalytic components of the RNA-induced silencing complex and play central roles in RNA interference. Using a combination of bioinformatics and rapid amplification of cDNA ends (RACE) methods, putative AGO subfamily members, ls-AGO1 and ls-AGO2, were cloned and characterized from the small brown planthopper, Laodelphax striatellus. The open reading frame (ORF) of ls-AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, and ls-AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. The expected conserved PAZ and PIWI domains, and the conserved Asp-Asp-His (DDH) catalytic triad motif in the PIWI domain were observed in both ls-AGO1 and ls-AGO2. Reverse transcription-qPCR (RT-qPCR) results showed that both ls-AGO1 and ls-AGO2 were expressed in all developmental stages of L. striatellus with highest mRNA abundance in eggs. Expression of ls-AGO1 and ls-AGO2 was significantly decreased in adult insects in response to acquisition of rice black-streaked dwarf virus by second instar nymphs. mRNA expression of ls-AGO1 was significantly downregulated in response to low and high temperatures, but expression of ls-AGO2 was only affected by low temperature. ls-AGO1 and ls-AGO2 were initially downregulated when insects were transferred from rice to maize and to the wild grass Brachypodium distachyon, but expression showed partial or complete recovery 7 days after transfer. These results document that AGO subfamily members of L. striatellus are ubiquitously expressed at different developmental stages and respond to various stresses. Thus, AGO subfamily may act in regulating the stress-response of L. striatellus by controlling related gene expression.

Abscisic acid negatively modulates plant defence against rice black-streaked dwarf virus infection by suppressing the jasmonate pathway and regulating reactive oxygen species levels in rice: A negative role of ABA in rice response to RBSDV

Abscisic acid (ABA) plays a multifaceted role in plant immunity and can either increase resistance or increase susceptibility to some bacterial and fungal pathogens depending on the pathosystem. ABA is also known to mediate plant defence to some viruses. In this study, the relationship between the ABA pathway and rice black-streaked dwarf virus (RBSDV) was investigated in rice. The expression of ABA pathway genes was significantly reduced upon RBSDV infection. Application of exogenous hormones and various ABA pathway mutants revealed that the ABA pathway plays a negative role in rice defence against RBSDV. Exogenous hormone treatment and virus inoculation showed that ABA inhibits the jasmonate-mediated resistance to RBSDV. ABA treatment also suppressed accumulation of reactive oxygen species by inducing the expression of superoxidase dismutases and catalases. Thus, ABA modulates the rice-RBSDV interaction by suppressing the jasmonate pathway and regulating reactive oxygen species levels. This is the first example of ABA increasing susceptibility to a plant virus.

Differential Characteristics of Viral siRNAs between Leaves and Roots of Wheat Plants Naturally Infected with Wheat Yellow Mosaic Virus, a Soil-Borne Virus

RNA silencing is an important innate antiviral defense in plants. Soil-borne plant viruses naturally infect roots via soil-inhabiting vectors, but it is unclear how antiviral RNA silencing responds to virus infection in this particular tissue. In this study, viral small interfering RNA (siRNA) profiles from leaves and roots of wheat plants naturally infected with a soil-borne virus, wheat yellow mosaic virus (WYMV, genus Bymovirus), were analyzed by deep sequencing. WYMV siRNAs were much more abundant in roots than leaves, which was positively correlated with the accumulation of viral RNA. WYMV siRNAs in leaves and roots were predominantly 21- and 22-nt long and equally derived from the positive- and negative-strands of the viral genome. WYMV siRNAs from leaves and roots differed in distribution pattern along the viral genome. Interestingly, compared to siRNAs from leaves (and most other reports), those from roots obviously had a lower A/U bias at the 5′-terminal nucleotide. Moreover, the expression of Dicer-like genes upon WYMV infection were differently regulated between leaves and roots. Our data suggest that RNA silencing in roots may operate differently than in leaves against soil-borne virus invasion.

AtHB2, a class II HD-ZIP protein, negatively regulates the expression of CsANS, which encodes a key enzyme in Camellia sinensis catechin biosynthesis

Tea (Camellia sinensis) is an important cash crop that is beneficial to human health because of its remarkable content of catechins. The biosynthesis of catechins follows the flavonoid pathway, which is highly branched. Among the enzymes involved in catechin biosynthesis, ANTHOCYANIDIN SYNTHASE (CsANS) functions at a branch point and play a critical role. Our previous work has showed that the gene encoding CsANS is regulated by light signals; however, the molecular mechanism behind remains unclear. Here, we cloned a full-length CsANS promoter and found that it contained a cis-element recognized by Arabidopsis thaliana HOMEOBOX2 (AtHB2). AtHB2 constitutes one of the class II HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) proteins, which accumulate in the dark and mediate the shade avoidance response in most angiosperms. To analyze the transcription of CsANS in vivo, β-glucuronidase and luciferase reporter genes driven by the obtained promoter were introduced into A. thaliana and Nicotiana attenuata, respectively. In both expression systems there were indications that the A. thaliana PRODUCTION OF ANTHOCYANIN PIGMENT1 (AtPAP1), a MYB transcription factor of flavonoid biosynthesis, increased the activity of the CsANS promoter, while AtHB2 could significantly undermine the effect of AtPAP1. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that AtHB2 interacted with the A. thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1). A yeast three-hybrid assay further suggested that AtHB2 represses the expression of CsANS and regulates its response to light signals through competitive interactions with AtTTG1. These results show that HD-ZIP II proteins participate in light regulation of flavonoid biosynthesis.

Ribotypes of Polymyxa graminis in wheat samples infected with soil-borne wheat viruses in China

Polymyxa graminis is an obligate parasite and important vector of more than 14 soilborne plant viruses that pose a significant threat to cereal crops in Europe, North America, and Asia. Different ribotypes or formae speciales of P. graminis have been recognized and these may be associated with different cereal hosts or with transmission of different viruses. Two soilborne viruses infecting winter wheat in China have been reported and well studied (Wheat yellow mosaic virus [WYMV, genus Bymovirus] and Chinese wheat mosaic virus [CWMV, genus Furovirus]) but there has been no reported characterization of P. graminis isolates associated with them. In this study, the ribosomal DNA internal transcribed spacer (ITS) regions of P. graminis were examined from 63 wheat samples with apparent virus symptoms obtained from 16 sites within six Chinese provinces. Their associations with soilborne viruses were investigated. Ribotype I (P. graminis f. sp. temperata) and ribotype II (P. graminis f. sp. tepida) were confirmed in winter wheat regions of China for the first time. All 63 wheat root samples were infected with ribotype I of P. graminis and 11 were also infected with ribotype II. There was no obvious association between the ribotypes and infection by either WYMV or CWMV (or double infection). Phylogenetic analysis of the P. graminis ITS1-5.8S-ITS2 sequences revealed that ribotype I in China belongs to previously reported subgroup Ib, whereas ribotype II belongs to IIa. There was considerable sequence variation (pairwise distances from 0.0219 to 0.0319) between Chinese ribotype I isolates of different regions and previously reported ribotype I isolate Ken5 (accession number HE860055.1).