CRISPR/Cas9-Mediated triple gene editing of Pi21, Bsr-d1, and Xa5 confers broad-spectrum disease resistance in elite early-season rice without yield compromise.

Rice blast and bacterial blight are devastating diseases that severely threaten global rice production. To develop durable resistance without compromising yield, we utilized CRISPR/Cas9 gene editing to simultaneously knock out three negative immune regulators, Pi21, Bsr-d1, and Xa5, in two elite early-rice cultivars (ZZU53 and ZZU100). We successfully obtained transgene-free homozygous triple mutants that exhibited significantly enhanced resistance to both Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). This broad-spectrum resistance correlates with the constitutive upregulation of key defense genes in the salicylic acid (SA) pathway (OsPR1a, OsPR1b, and OsWRKY45) and jasmonic acid (JA) pathways (OsPR4). Comprehensive agronomic evaluation demonstrated that critical agricultural traits such as thousand-grain weight, panicle architecture, heading date, and tiller number remained comparable to wild-type plants, indicating no substantial yield penalty. This approach provides an efficient, transgene-free pathway for developing disease-resistant early-rice cultivars. Multiple environment field trials are now required to evaluate its long-term agricultural viability.