Virus inoculation is a basic experimental procedure to evaluate the resistance of a rice variety or a transgenic material upon virus infection. We recently demonstrated that Rice Ragged Stunt Virus (RRSV), an oryzavirus that is transmitted by brown planthopper (BPH), can suppress jasmonic acid-mediated antiviral defense through the induction of microRNA319 and facilitate virus infection in rice. To verify this, we performed virus inoculation experiments on wild-type rice plants and miR319-TCP21-associated transgenic rice plants through a modified group inoculation method. Here, we presented the detailed procedure of RRSV propagation and infection process on rice plants.

Fusarium graminearum, the major causal agent of Fusarium head blight (FHB), causes serious wheat yield losses and a threat to human and animal health. The main efforts to combat the disease are the research of pathogenesis mechanisms and breeding for disease resistance plants. The efficiency of these actions could be evaluated by reliable inoculation assay, which is performed by accurate and repeatable inoculation methods. Hence, a standard procedure of effective wheat inoculation should improve the accuracy of pathogenicity evaluation. Here, we present a protocol for wheat spike inoculation with fungal conidial suspensions or fungus agar discs. These methods show highly reproducibility and accuracy on wheat infection experiment in laboratory conditions.

Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation – one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).

Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.

Virus-based microRNA silencing (VbMS) is a viable and prompt method to screen and characterize the function of microRNAs (miRNAs) in plants. The Tobacco rattle virus (TRV)-based VbMS method was originally developed by the Yule Liu's group (Sha et al., 2014) using miRNA target mimic (TM) methodology. Here, we describe the TRV-based VbMS method for silencing endogenous miRNA in Nicotiana benthamiana and tomato via Agrobacterium infiltrations. For each assay, Agrobacterium cultures containing pTRV1 and specific pTRV2e derivative harboring TM fragments are mixed and infiltrated into plant tissues. Generally within 3 weeks, the target miRNAs gene will be silenced and the newly developed tissues will exhibit corresponding phenotypes.

This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris et al., 2013). It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. It is adapted from the calculations described by (Strain et al., 1971).

microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.

Virus-induced gene silencing (VIGS) is a powerful method to study gene function in plants. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector so far. This method was originally developed by the Dinesh-Kumar's group (Liu et al., 2002) . Here, we describe a rapid and high efficient TRV-based VIGS method for knocking down genes in Nicotiana benthamiana. For TRV-based VIGS, Agrobacterium culture containing pTRV1 and Agrobacterium culture containing pTRV2 with plant target gene fragment are mixed and infiltrated into the lower leaves of plant. After 2-3 weeks post infiltration, plant target gene will be silenced.

DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.