Journal: The EMBO Journal

Article Title: A double-agent microRNA regulates viral cross-kingdom infection in animals and plants

doi: 10.1038/s44318-025-00405-4

Figure Lengend Snippet: ( A ) Comparative plant height between WT and OE263a lines, with a scale bar representing 50 cm. Twelve biological replicates were prepared, with each replicate including one rice plant. ( B ) Plant height comparison between WT and GATA19 KO lines, utilizing the same scale bar for reference. Twelve biological replicates were prepared, with each replicate including one rice plant. ( C ) The 1000-grain weight of WT and OE263a lines. Six biological replicates were prepared. ( D ) The 1000-grain weight of WT and GATA19 KO lines. Six biological replicates were prepared. ( E ) Husked grain width and length of WT, OE263a, and GATA19 KO lines. Scale bars: 5 mm. Five to six biological replicates were prepared, with each replicate including 10 grains. ( F ) Disease symptom of WT, OE263a, and GATA19 KO lines after inoculation with RSV. For ( A ) to ( E ), the values are reported as mean ± SE. Data comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s test. Different letters indicate significant differences.

Article Snippet: Rice proteins including JAZ1, JAZ10, GATA19, JAZ8, and MYC2 were synthesized using Premium ONE Expression Kit (Tiangen; CFS-EDX-ONE) following the manufacture’s protocol.

Techniques: Comparison

Journal: The EMBO Journal

Article Title: A double-agent microRNA regulates viral cross-kingdom infection in animals and plants

doi: 10.1038/s44318-025-00405-4

Figure Lengend Snippet: ( A ) Relative transcript level of GATA19 and its primary transcript (Pri-GATA19) in WT and OE263a rice lines. ( B ) Generation of GATA19 knockout ( GATA19 KO) mutant rice lines via CRISPR/Cas9: On the top, the placement of two sgRNAs (denoted by pink circles) is illustrated within GATA19 ’s open reading frame. The red arrow signifies the experimentally validated splicing site, confirmed through 5′RLM-RACE and sequencing, with the adjacent number reflecting the frequency of 5′RLM-RACE products cleaved at that specific site. Turning to the bottom, the diagram showcases the two sgRNA sequences that specifically target GATA19 , with a pink arrow pinpointing the site of induced mutation. ( C ) miRNA target-GFP reporter expression assay for the interaction of miR-263a and the target site within the first intron of GATA19 . Rice protoplasts were cotransfected with pBI-EGFP-target and pBI-OE263a, or cotransfected with pBI-EGFP-target and pBI221 empty vectors. Images were captured at 36 h post-transfection. Scale bars: 5 μm. Relative transcript level of miR-263a in rice protoplasts was detected by qPCR. P = 0.0065. ( D ) Relative transcript levels of mRNA from an artificial primary transcript of GATA19 and miR-263a measured in rice protoplasts. Protoplasts were transfected with a recombinant pBI221 plasmid containing the first two exons and the intervening intron of GATA19 ( Ex-In-Ex ) or a mutant plasmid with a 17-nucleotide mutated target site within the intron ( Ex-InMT-Ex ). Transfections were conducted either in combination with the miR-263a overexpression vector (OE263a) or with the empty pBI221 vector as a control. ( E ) Protein analysis of GATA19 in WT and GATA19 KO mutant lines using Western blot assays. NP was detected using a homemade monoclonal anti-NP antibody. Plant Actin that was detected using a monoclonal anti-Actin antibody was used as the reference protein. The relative optical densities of NP to that of Actin were calculated. ( F ) Relative RNA level of viral NP in GATA19 KO mutant lines compared to WT post inoculation with viruliferous fourth-instar SBPH larvae. ( G ) The disease incidence of WT and GATA19 KO rice fed viruliferous fourth-instar SBPH larvae for 7 d. Five rice seedlings per replicate and six replicates were applied. P = 0.0100 (13 dpi), P = 0.0031 (14 dpi), P = 0.0065 (15 dpi), P = 0.0058 (16 dpi), P = 0.0310 (20 dpi), P = 0.0351 (24 dpi), P = 0.0299 (25–30 dpi), from left to right. For ( A ), ( D ), and ( F ), data are shown as mean ± SE. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s test. Different letters indicate significant differences. For ( C ) and ( G ), values are shown as mean ± SE and were compared by Student’s t test. * P < 0.05. ** P < 0.01. .

Article Snippet: Rice proteins including JAZ1, JAZ10, GATA19, JAZ8, and MYC2 were synthesized using Premium ONE Expression Kit (Tiangen; CFS-EDX-ONE) following the manufacture’s protocol.

Techniques: Knock-Out, Mutagenesis, CRISPR, Sequencing, Expressing, Transfection, Recombinant, Plasmid Preparation, Over Expression, Control, Western Blot

Journal: The EMBO Journal

Article Title: A double-agent microRNA regulates viral cross-kingdom infection in animals and plants

doi: 10.1038/s44318-025-00405-4

Figure Lengend Snippet: ( A ) Quantification of JA and JA-Ile in WT, OE263a, and GATA19 KO lines. Four biological replicates were prepared. Data are shown as mean ± SE. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s or Duncan’s test. Different letters indicate significant differences. ( B ) Quantification of JA and JA-Ile in WT rice fed upon by nonviruliferous SBPHs with injection of antagomir-263a or NC. Three biological replicates were prepared . P = 0.0015. Values are shown as mean ± SE and were compared by Student’s t test. NS, no significant difference. ** P < 0.01. ( C ) Recombinantly expressed JAZ1/JAZ8/JAZ10-His binds the recombinantly expressed GATA19-V5 in the Co-IP and Western blot assay. Mouse IgG was used as a negative control. ( D ) Recombinantly expressed JAZ1-His binds the GATA19 from rice nuclear extracts in the Co-IP and Western blot assay. ( E ) BiFC assay for the interaction of JAZ1-YC with GATA19-YN in Nicotiana benthamiana epidermal cells. Images were captured at 36 h post-infiltration. Scale bars: 20 μm. ( F ) Recombinantly expressed GATA19-V5 binds the recombinantly expressed MYC2-Flag in the Co-IP and Western blot assay. ( G ) His-tag pull-down assay for the competitive binding of JAZ1-His by GATA19-V5 and MYC2-Flag using recombinantly expressed proteins. The GATA19-V5 recombinant protein was incubated with JAZ1-His at varying concentrations, specifically at ratios of 1:50:500. .

Article Snippet: Rice proteins including JAZ1, JAZ10, GATA19, JAZ8, and MYC2 were synthesized using Premium ONE Expression Kit (Tiangen; CFS-EDX-ONE) following the manufacture’s protocol.

Techniques: Injection, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Bimolecular Fluorescence Complementation Assay, Pull Down Assay, Binding Assay, Recombinant, Incubation

Journal: The EMBO Journal

Article Title: A double-agent microRNA regulates viral cross-kingdom infection in animals and plants

doi: 10.1038/s44318-025-00405-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rice proteins including JAZ1, JAZ10, GATA19, JAZ8, and MYC2 were synthesized using Premium ONE Expression Kit (Tiangen; CFS-EDX-ONE) following the manufacture’s protocol.

Techniques: Virus, Recombinant, FLAG-tag, Sequencing, Labeling, Protease Inhibitor, Reverse Transcription, Membrane, Transfection, Hybridization, Blocking Assay, Caspase-3 Activity Assay, Activity Assay, TUNEL Assay, Apoptosis Assay, Fluorescence, Luciferase, SYBR Green Assay, Expressing, Cloning, Extraction, Transformation Assay, Immunoprecipitation, Software