Abstract

MicroRNAs play crucial roles in plant growth, development, and disease resistance. Transcriptome screening previously identified three late blight-responsive miRNAs (sly-miR172a, sly-miR156e, and sly-miR395a) in tomato whose biological functions remain uncharacterized. Here, CRISPR/Cas9 editing vectors targeting each miRNA were transformed into the tomato cultivar ‘Micro-Tom’. sly-miR395a showed the highest T0 editing efficiency (77.78%), followed by sly-miR172a (48.00%) and sly-miR156e (40.74%). T1 segregation ratios were approximately 1:12.3, 1:1.83, and 1:13 for the three targets. Stable T2 homozygous lines were obtained, and qRT-PCR confirmed average mature miRNA inhibition rates of 78.00%, 79.50%, and 87.50%. To assess editing specificity, genome-wide off-target prediction and Sanger sequencing of top high-risk loci were performed; no unintended indels were detected, confirming sgRNA specificity. Agronomic phenotyping revealed divergent developmental phenotypes: sly-miR172a knockout caused dwarfism, leaf senescence and reduced flower number; sly-miR156e mutants exhibited taller plants and increased floral production; sly-miR395a mutants had normal vegetative growth but fewer flowers. Resistance assays demonstrated distinct responses to Phytophthora infestans. Loss of sly-miR172a increased pathogen biomass by 1.6–2.3-fold and aggravated susceptibility, likely by de-repressing AP2/TOE to disrupt oxidative homeostasis. In contrast, both sly-miR156e and sly-miR395a negatively regulate late blight resistance. This could be because sly-miR156e knockout relieved the repression of SPL transcription factors, significantly restricting pathogen proliferation and lesion formation, and notably broke the canonical plant growth–defense trade-off to achieve simultaneous improvement of agronomic traits and disease resistance. sly-miR395a knockout enhanced tomato immunity potentially by activating sulfur metabolism-related pathways to accumulate defensive metabolites, with negligible adverse effects on vegetative growth. Nevertheless, this study has several limitations: the proposed regulatory cascades, including miRNA–AP2, miRNA–SPL, and miRNA–sulfur metabolism modules remain hypothetical without direct molecular validation of target interactions. Collectively, this study genetically validates that sly-miR172a, sly-miR156e, and sly-miR395a exert divergent functions in regulating tomato growth and immunity against late blight, providing valuable germplasm and gene resources for tomato disease-resistance breeding.

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