Figure 5

Rice sRNA biogenesis genes impact xisRNA accumulation. The images of autoradiographs obtained after RNA gel northern blot analysis conducted with specific oligonucleotide probes hybridized to total RNA extracted from leaves of wild type Nipponbare (WT) or a OsDCL1-silenced line (DCL1-IR) in (A), wild type Kinmaze (WT) or a OsHEN1 mutant allele line (waf1-2) in (B) or Nipponbare in (C). Leaves were infiltrated with the indicated strains and collected 24 hpi. These results are representative of two (OsDCL1) and three (OsHEN1 and Tal1c blots) replicate experiments. In (C), the plot on top of the gel images represent OsHEN1 Q-RT-PCR expression data from four experiments (light gray point) with mean (dark gray points) and standard error (line ranges). The BLS256H strains correspond to a T3SS minus mutant derivative of BLS256. The “+” sign indicate that the strains were transformed with a plasmid expressing Tal1c or the corresponding empty vector (EV). Full-length blots are presented in Supplementary Fig. S15. (D) Diagram depicting the general exon–intron structure of OsDCL4 and a close-up genome browser view of the last exon of the gene with the 3′ UTR. The dcl4-1 deletion is not drawn to scale but span ~ 1.5 kb on the 5′-side of the gene which includes upstream promoter sequences up to the first 72 bp of exon 1²⁷. The genome view represents reads coverage in various treatments, the MSU7 annotation, and the xisRNA022 locus defined in the “BAI3” and “Diversity” sRNA-seq datasets as well as its duplex locus. For details see the legend of Fig. 1F. (E) Northern blot analysis conducted on total RNA extracted at 48 hpi from leaves of homozygous wild type (WT) or homozygous dcl4-1 individuals. This experiment was repeated three times with similar results. In (A), (B) and (E), lanes with the same labels correspond to biological replicates of total RNA samples extracted from a single infiltrated leaf area from individual independent plants. The hyphens on the right of individual blots mark a size of 21 nt. For each panel, individual blot images derive from successive stripping, hybridization and detection rounds of the same membrane.