Ran, Danlu

Abstract The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.

AbstractCitrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CaLas), is the most serious disease worldwide. CaLasSDE460 was previously characterized as a potential virulence factor of CaLas. However, the function and mechanism of CaLasSDE460 involved in CaLas against citrus is still elusive. Here, we showed that transgenic expression of CaLasSDE460 in Wanjincheng oranges (C. sinensis Osbeck) contributed to the early growth of CaLas and the development of symptoms. When the temperature increased from 25 °C to 32 °C, CaLas growth and symptom development in transgenic plants were slower than those in WT controls. RNA-seq analysis of transgenic plants showed that CaLasSDE460 affected multiple biological processes. At 25 °C, transcription activities of the “Protein processing in endoplasmic reticulum” and “Cyanoamino acid metabolism” pathways increased while transcription activities of many pathways decreased at 32 °C. 124 and 53 genes, separately annotated to plant-pathogen interaction and MAPK signaling pathways, showed decreased expression at 32 °C, compared with these (38 for plant-pathogen interaction and 17 for MAPK signaling) at 25 °C. Several important genes (MAPKKK14, HSP70b, NCED3 and WRKY33), remarkably affected by CaLasSDE460, were identified. Totally, our data suggested that CaLasSDE460 participated in the pathogenesis of CaLas through interfering transcription activities of citrus defense response and this interfering was temperature-dependent. Graphical Abstract