A Significantly High Abundance of “Candidatus Liberibacter asiaticus” in Citrus Fruit Pith: in planta Transcriptome and Anatomical Analyses

Abstract

Huanglongbing, a highly destructive disease of citrus, is associated with the non-culturable phloem-limited α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). The distribution patterns of CLas in infected plant are variable and not consistent, which make the CLas detection and characterization more challenging. Here, we performed a systemic analysis of CLas distribution in citrus branches and fruits of 14 cultivars. A significantly high concentration of CLas was detected in fruit pith (dorsal vascular bundle) of 14 citrus cultivars collected at fruit maturity season. A 2-year monitoring assay of CLas population in citrus branches of “Shatangju” mandarin (Citrus reticulata Blanco “Shatangju”) revealed that CLas population already exhibited a high level even before the appearance of visual symptoms in the fruit rind. Quantitative analyses of CLas in serial 1.5-cm segments of fruit piths showed the CLas was unevenly distributed within fruit pith and tended to colonize in the middle or distal (stylar end) regions of pith. The use of CLas-abundant fruit pith for dual RNA-seq generated higher-resolution CLas transcriptome data compared with the leaf samples. CLas genes involved in transport system, flagellar assembly, lipopolysaccharide biosynthesis, virulence, stress response, and cell surface structure, as well as host genes involved in biosynthesis of antimicrobial-associated secondary metabolites, was up-regulated in leaf midribs compared with fruit pith. In addition, CLas infection caused the severe collapse in phloem and callose deposition in the plasmodesmata of fruit pith. The ability of fruit pith to support multiplication of CLas to high levels makes it an ideal host tissue for morphological studies and in planta transcriptome analyses of CLas–host interactions.

An Overview of the Mechanisms Against “Candidatus Liberibacter asiaticus”: Virulence Targets, Citrus Defenses, and Microbiome

“…In vivo transcriptomic analyses are required to understand the active pathways in C Las. A recent study identified the regions in the citrus fruit pith with higher bacterial titers which was used to conduct RNA-seq analysis after rRNA removal ( Fang et al, 2021 ). This study compared the gene expression profiles of the fruit pit vs. leaf midribs and found different gene expression profiles related to virulence genes; however, the resolution of the transcriptome profile was lower in midribs compared to fruit pit mainly due to the lower bacterial titers ( Fang et al, 2021 ).…”

Molecular signatures between citrus and Candidatus Liberibacter asiaticus

“…Host responses against C Las infections vary in different tissues of same plants. The virulence, stress response, and antimicrobial secondary metabolites-associated genes show enhanced expression in midrib tissues from the leaves compared to the fruit piths in HLB-positive Citrus reticulata Blanco “Shatangju” [ 150 ]. Similarly, the temporal host response may vary in citrus against C Las infestation.…”

Biological Features and In Planta Transcriptomic Analyses of a Microviridae Phage (CLasMV1) in “Candidatus Liberibacter asiaticus”

“…), a holoparasitic plant, from one infected citrus plant to other citrus plants or other amenable hosts, e.g., periwinkle ( Catharanthus roseus (L.) G. Don) [ 2 ]. CLas can infect most commercial citrus cultivars and present as an uneven distribution pattern within infected citrus plants [ 3 , 4 ]. The distribution pattern of CLas in infected citrus trees could be different, mainly due to the different cultivars and degrees of disease severity [ 5 , 6 ].…”

“…The distribution pattern of CLas in infected citrus trees could be different, mainly due to the different cultivars and degrees of disease severity [ 5 , 6 ]. However, an investigation of CLas distribution in 14 commercial citrus cultivars showed that a significantly high abundance of CLas was stably observed in fruit pith tissue compared to other tissues from the individual citrus branch [ 4 ].…”