Two conserved early effectors from Puccinia triticina are required for pathogenicity by suppressing callose deposition and hydrogen peroxide accumulation in wheat

BACKGROUND: Wheat leaf rust, caused by the causal agent Puccinia triticina (Pt), poses a serious threat to wheat production worldwide. Pt secretes plenty of effectors into the host plant to manipulate host immune responses and facilitate successful colonization. However, effectors during the early stages of Pt infection remain largely unknown. RESULTS: Here, we employed deep RNA-sequencing on samples extracted from wheat leaves of a susceptible wheat cultivar, Thatcher, inoculated with Pt at 8 and 24 h post-inoculation, which enabled the identification of candidate effectors implicated in suppressing early plant immune responses. Through integrated transcriptomic analysis and an optimized effector identification workflow, early candidate effector-encoding genes that are highly expressed during Pt infection were identified and characterized. Functional assays revealed that two effector candidates, Pt01364 and Pt09156, exhibited the ability to suppress Bax-induced cell death in Nicotiana benthamiana. Quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that both genes peaked at 8 h post-inoculation, and the yeast secretion trap assay showed that these two cell death-suppressing effector candidates possess functional signal peptides for secretion into host cells. Silencing of Pt01364 and Pt09156 significantly compromised Pt pathogenicity on a susceptible wheat cultivar. In addition, both effectors can inhibit callose deposition triggered by non-host bacteria and reduce hydrogen peroxide accumulation in wheat in response to avirulent Pt infection. CONCLUSION: This study provides a basis for identifying key effectors involved in the early stages of wheat-Pt interaction, highlighting Pt01364 and Pt09156 as promising candidate targets for developing innovative strategies to control wheat leaf rust. © 2026 Society of Chemical Industry.