Ghosh, Dilip Kumar

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.

The amino acid‐binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine‐binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine‐binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand‐bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C‐terminal extended loop of one chain very close to the substrate‐binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C‐terminal extended loop in position. Analysis of the substrate‐binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg‐bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix‐α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand‐free open state. The dissociation constant of 1.26 μm by SPR and 3.7 μm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l‐cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.