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.

Keywords: Candidatus Liberibacter asiaticus; anatomical analyses; citrus huanglongbing; fruit pith; high abundance; in planta transcriptome.

FIGURE 1

Quantitative distribution off “Candidatusf Liberibacter asiaticus” (CLas) in citrus branches and fruits. (A) Symptom development of HLB-affected “Shatangju” mandarin (Citrus reticulata Blanco “Shatangju”) fruits. (B) Temporal dynamics of CLas population in different parts of HLB-affected “Shatangju” branches. Ten citrus branches were monthly collected from HLB-affected citrus trees during August 15, 2017, to January 15, 2018, and August 15, 2018 to January 15, 2019. Note the vascular columella (white arrow) under the peduncle of diseased fruit started to turn into orange in October.

FIGURE 2

Quantitative distribution of “Candidatus Liberibacter asiaticus” (CLas) in fruit piths of three citrus varieties. Graphic denoted 1.5-cm segment of fruit piths. Color scale indicates the CLas cell number in each 1.5-cm segment following quantitative real-time PCR. The average number of CLas cells in each 1.5-cm segment of fruit piths from individual citrus variety is indicated as bar graph. The different letters at the top of the bar represent significant difference by single-factor analysis of variance (Duncan multiple-range test) at 95% (P < 0.05) confidence interval.

FIGURE 3

Functional classification of differentially expressed genes (A) and top 20 highly expressed gene (B) of “Candidatus Liberibacter asiaticus” (CLas) in fruit pith and leaf midribs. The differentially expression genes (DEGs) are identified with GFOLD v1.1.4 software by setting log2 fold change ≥ | 1 |. Classification of DEGs is performed with EggNOG v5.0 by using Clusters of Orthologous Groups database. Black bar represents genes up-regulated in fruit pith compared with leaf midribs. Gray bar represents genes up-regulated in leaf midribs compared with fruit pith. Heatmap shows the log2 of normalized TPM values for the top 20 most highly expressed CLas genes in leaf midribs tissue and fruit pith tissue. Genes that belong to the list of the top 20 most highly expressed genes in leaf midribs or fruit piths are labeled with leaf or fruit symbol in the left.

FIGURE 4

Volcano plot representation of differential expression analysis of host genes in “Candidatus Liberibacter asiaticus” (CLas)–infected leaf midribs and CLas-infected fruit pith. Red and green points mark the genes with significantly increased or decreased expression, respectively, in leaf midribs compared with fruit pith samples (FDR < 0.01). The blue points mark the genes with no significantly different expression. The x-axis represents log2 fold changes in expression, and the y-axis represents statistically significant difference in gene expression.

FIGURE 5

GO term enrichment analyses of the up-regulated genes in “Candidatus Liberibacter asiaticus” (CLas)–infected fruit pith (A) and CLas-infected leaf midribs (B). The vertical coordinates represent enriched GO terms, and the horizontal coordinates represent the numbers of up-regulated genes in these GO terms. Green columns: biological process GO terms; purple columns: cellular component GO terms; orange columns: molecular function GO terms. *Significantly enriched GO term.

FIGURE 6

KEGG enrichment analyses of the up-regulated genes in “Candidatus Liberibacter asiaticus” (CLas)–infected leaf midribs (A) and CLas-infected fruit pith (B). The vertical coordinates represent the enriched pathways, and the horizontal coordinates represent the rich factors. The size of each point represents the number of up-regulated genes in the pathway, and the color of the point represents the q value.

FIGURE 7

Light micrographs of leaf midribs and fruit pith from “Candidatus Liberibacter asiaticus”–infected and healthy “Gongkan” and “Shatangju” mandarin. X, xylem; F, fiber; P, phloem. The callose plugs (yellow arrow), collapsed phloem (black arrow), and starch granule accumulation (white arrow) are commonly observed in CLas-infected lead midrib. Bar = 30 μm.

FIGURE 8

Transmission electron micrographs of cross section of fruit pith tissues from “Candidatus Liberibacter asiaticus”–infected “Shatangju” mandarin. (A) Collapsed phloem cells and callose deposition in the plasmodesmata pore between sieve elements of CLas-infected fruit pith. (B) A high abundance of CLas cells with elongated and round forms in the phloem cell. (C) The close-up of red region in panel (B). SE, sieve element; Ca, callose; FM, filamentous material found around CLas cells. *Phloem cells collapse. Note double cell walls (white arrows) characteristic of Gram-negative bacterium, which is presumed to be CLas cells.