Publications

4533

Abstract Candidatus Liberibacter asiaticus ( C Las), the causal agent of Huanglongbing, is transmitted by the Asian citrus psyllid ( Diaphorina citri ) in a persistent, propagative manner. Reproducible transmission studies require not only controlled inoculation assays but also standardized propagation of citrus host plants, maintenance of D. citri colonies, and reliable pathogen detection. This study presents a comprehensive set of coordinated protocols that collectively support controlled C Las transmission experimentation. The workflow includes propagation of Citrus macrophylla for colony and assay use; generation and maintenance of C Las‐infected citrus and D. citri colonies; synchronization of D. citri populations to enable C Las acquisition; and a controlled single‐ D. citri transmission assay with defined inoculation access periods, and a Dellaporta‐based nucleic acid extraction method compatible with citrus and psyllid tissues. Quantitative PCR detection is integrated throughout to verify infection status, determine C Las titer, and monitor C Las movement and systemic colonization in citrus over time. By combining standardized plant production, vector infection systems, transmission assays, and molecular diagnostics, these coordinated methods provide a unified framework for testing hypotheses about vector competence, pathogen–vector interactions, microbial co‐infections, host susceptibility, and environmental effects on transmission. Together, the procedures enable consistent and comparable assessment of C Las acquisition, dissemination, and transmission outcomes under controlled laboratory conditions. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Controlled laboratory method for assessing Candidatus Liberibacter asiaticus transmission by Diaphorina citri Support Protocol 1 : Generation of Candidatus Liberibacter asiaticus‐infected Citrus macrophylla and Diaphorina citri Basic Protocol 2 : Dellaporta total nucleic acid extraction Basic Protocol 3 : Citrus macrophylla propagation

ABSTRACT Citrus Huanglongbing (HLB), caused by ‘ Candidatus Liberibacter asiaticus’ ( Ca Las), is the most devastating disease affecting the global citrus industry. Here, we reported that the Ca Las effector SDE70 promotes HLB pathogenicity by targeting the citrus ubiquitination pathway. Transgenic expression of SDE70 in Wanjincheng orange ( Citrus sinensis Osbeck) accelerated early Ca Las proliferation, aggravated HLB symptoms, and increased susceptibility to citrus canker induced by Xanthomonas citri subsp. citri ( Xcc ). These results demonstrate that SDE70 functions as a broad‐spectrum suppressor of citrus immunity. Mechanistically, SDE70 physically interacts with CsRUB2, a citrus ubiquitin‐related protein. Furthermore, CsRUB2 overexpression in Wanjincheng oranges reduced resistance to HLB but enhanced resistance to citrus canker. Both SDE70 and CsRUB2 elevated salicylic acid (SA) and hydrogen peroxide (H 2 O 2 ) levels in transgenic plants while lowering methyl salicylate (MeSA) levels. CsRUB2 also decreased jasmonic acid (JA). In contrast to the suppressive effect of SDE70, CsRUB2 enhanced the transcription of citrus immunity genes. Transient expression assays further demonstrated that the SDE70–CsRUB2 interaction dysregulates citrus immunity by perturbing SA, MeSA, JA, and H 2 O 2 signals. These findings provide a theoretical basis for understanding citrus– Ca Las interactions and breeding citrus varieties with broad‐spectrum resistance to both HLB and citrus canker.