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Summary  Huanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium,  Candidatus  Liberibacter asiaticus ( C Las), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB‐tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB‐tolerant citrus hybrid ( Poncirus trifoliata  ×  Citrus reticulata ) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB‐tolerance. These regulators display similar expression patterns in another HLB‐tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid ( Microcitrus virgata ). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a  C . Liberibacter solanacearum ( C Lso ) /potato psyllid/ Nicotiana benthamiana  interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus‐induced gene silencing in  N .  benthamiana , this innovative and cost‐effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1‐Associated Protein, and the negative regulator Vascular Associated Death Protein.

Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system