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: miRcute Plus miRNA First-Strand cDNA Kit , Tiangen , Cat# 4992909.

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: OsU6 snRNA and UBQ10 serve as internal references for miRNA and gene, respectively. Twelve biological replicates were prepared, with each replicate including two leaves. P values from left to right, P = 0.0202, P = 0.0211. Values are presented as mean ± SE and were compared by Student’s t test. * P < 0.05.

Article Snippet: miRcute Plus miRNA First-Strand cDNA Kit , Tiangen , Cat# 4992909.

Techniques:

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: miRcute Plus miRNA First-Strand cDNA Kit , Tiangen , Cat# 4992909.

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