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The common rust disease of maize is caused by the obligate biotrophic fungusPuccinia sorghi. The maizeRp1-Dallele imparts resistance against theP.sorghiIN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D fromP.sorghiIN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize andP.sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes,AvrRp1-D.1andAvrRp1-D.2, which triggered anRp1-D-dependent defense response inNicotiana benthamiana.AvrRp1-D-inducedRp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize.