Stage-Specific RNA Turnover Drives Small RNA Dynamics in Arabidopsis-Colletotrichum Interactions

Small RNAs (sRNAs) are key regulators of plant defense and have been implicated in cross-kingdom interactions with pathogens. The hemibiotrophic fungus Colletotrichum higginsianum infects Arabidopsis thaliana through three stages: appressorial penetration, biotrophy, and necrotrophy. However, the dynamics of fungal and plant sRNA populations across these three stages have not been elucidated. Using high-throughput sequencing, we profiled sRNAs from A. thaliana and C. higginsianum during in planta appressorium (PA), biotrophic (BP), and necrotrophic (NP) phases, and compared them to fungal mycelia (MY) and in vitro appressoria (VA). Our analyses revealed stage-specific patterns in sRNA accumulation in both the plant and the pathogen. In C. higginsianum, sRNAs were dominated by 29 nt species in PA, BP, MY, and VA, but shifted to 18 nt in NP, consistent with RNA degradation during host cell death. In A. thaliana, sRNAs transitioned from 30-33-nt in PA/BP to a 21 nt dominant peak in NP. Also, TE-derived siRNAs and other regulatory sRNAs (miRNAs, ncRNA, snoRNAs and snRNAs) declined during NP. A total of 62 host miRNAs showed differential accumulation, including core plant developmental regulators active across infection stages, and stage-specific miRNAs such as miR396, miR170/171, miR472, and miR858b. tRFs displayed opposite trends in host and pathogen: fungal tRFs declined in NP, while host misc-tRFs, 5'-tRFs, and 3'-tRFs increased, suggesting contrasting regulatory roles. These results provide new insights into RNA-mediated plant-fungal interactions.