RNAi-Mediated Attenuation of CsCalS7-Related Transcripts Mitigates Callose Accumulation and Huanglongbing Symptoms

Huanglongbing (HLB) is the most destructive citrus disease worldwide and is characterised by excessive callose deposition at sieve plates, which has been associated with impaired carbohydrate transport, starch accumulation and severe symptoms. CsCalS7, a phloem-associated callose synthase, is induced during infection and involved in sieve pore occlusion. Citrus sinensis contains at least three CalS7 genes (CsCalS7a, CsCalS7b and CsCalS7c). Here, RNA interference (RNAi) was employed to reduce the CsCalS7-related transcript levels. Similarity analyses showed the hairpin-RNAi (hpRNAi) perfectly matched CsCalS7a and shared similarity with CsCalS7b and CsCalS7c. siRNA-level analysis predicted strong silencing of CsCalS7a and possible moderate effects on CsCalS7b and CsCalS7c, supporting modulation of multiple paralogs. Reverse transcription-quantitative PCR confirmed partial transcript reduction (10%-62%) across independent lines, and microscopic analysis revealed a significant reduction of phloem callose deposition in RNAi lines. After 'Candidatus Liberibacter asiaticus' (CLas) inoculation, several RNAi lines showed reduced starch accumulation and attenuated HLB symptom severity compared to non-transgenic controls. Bacterial levels did not differ significantly among genotypes at 5 months post-inoculation in the evaluated plant tissues. Together, these results demonstrate that moderate suppression of CsCalS7-related transcripts reduces callose deposition and possibly mitigates starch overaccumulation and symptom severity. The observed effects of partial RNAi-mediated suppression suggest that modulating callose synthase activity can improve host tolerance to HLB.