Comparative Transcriptome and sRNAome Analysis Suggest Coordinated Citrus Immune Responses against Huanglongbing Disease.

Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Candidatus Liberibacter asiaticus ( C Las), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to C Las infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon ( Citrus limon L.) with HLB-susceptible Shatangju mandarin ( Citrus reticulata Blanco cv. Shatangju) against C Las infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, C Las infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like H 2 O 2 (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that C Las infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against C Las. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with C Las, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.