Transcriptional and microscopic analyses of citrus stem and root responses to Candidatus Liberibacter asiaticus infection

Abstract

Huanglongbing (HLB) is the most destructive disease that affects citrus worldwide. The disease has been associated with Candidatus Liberibacter. HLB diseased citrus plants develop a multitude of symptoms including zinc and copper deficiencies, blotchy mottle, corky veins, stunting, and twig dieback. Ca. L. asiaticus infection also seriously affects the roots. Previous study focused on gene expression of leaves and fruit to Ca. L. asiaticus infection. In this study, we compared the gene expression levels of stems and roots of healthy plants with those in Ca. L. asiaticus infected plants using microarrays. Affymetrix microarray analysis showed a total of 988 genes were significantly altered in expression, of which 885 were in the stems, and 111 in the roots. Of these, 551 and 56 were up-regulated, while 334 and 55 were down-regulated in the stem and root samples of HLB diseased trees compared to healthy plants, respectively. Dramatic differences in the transcriptional responses were observed between citrus stems and roots to Ca. L. asiaticus infection, with only 8 genes affected in both the roots and stems. The affected genes are involved in diverse cellular functions, including carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress responses, signaling and transcriptional factors, transportation, cell organization, protein modification and degradation, development, hormone signaling, metal handling, and redox. Microscopy analysis showed the depletion of starch in the roots of the infected plants but not in healthy plants. Collapse and thickening of cell walls were observed in HLB affected roots, but not as severe as in the stems. This study provides insight into the host response of the stems and roots to Ca. L. asiaticus infection.

Figure 1. Differential regulation of genes in the stems and roots of Valencia sweet orange (Citrus sinensis) by Ca. L. asiaticus infection.

Figures in parentheses indicate total numbers of genes in the stems and in the roots. The numbers of significantly up-regulated genes are shown in green, and down-regulated are shown in red. A total of 998 genes showed significantly altered expression in the stems and roots.

Figure 2. Overview of metabolic pathways that are regulated by Ca. L. asiaticus infection in the stems and roots of Valencia sweet orange (Citrus sinensis).

A = stem and B = root. Genes that were significantly up-regulated following Ca. L. asiaticus infection are displayed in blue, and down-regulated genes are displayed in red.

Figure 3. Regulation of biotic stress-related gene pathways by Ca. L. asiaticus infection in the stems and roots of Valencia sweet orange (Citrus sinensis).

A = stem and B = root. Genes that were significantly up-regulated following Ca. L. asiaticus infection are displayed in blue, and down-regulated genes are displayed in red.

Figure 4. Regulation of receptor-like kinase (RLK) genes by Ca. L. asiaticus infection in the stems of Valencia sweet orange (Citrus sinensis).

Genes that were significantly up-regulated following Ca. L. asiaticus infection are displayed in blue, and down-regulated genes are displayed in red. Abbreviations/definitions: LRR, leucine-rich repeats; Extensin, RLK with extensin motif; LysM, RLKs with lysine motif; C-lectin, RLKs with lectin-like motifs; Crinkly4-like, RLKs with crinkly4-like domains; DUF26, domain of unknown function 26; LRK 10-like, RLK gene linked to Lr10 locus; L-lectin, RLKs with lectin-binding domains; PERK-like, proline-rich extensin-like kinase; S-locus, RLK with S-domain similar to S-locus glycoproteins; RKF3-like, receptor-like kinase in flowers 3; Thaumatin, RLK-like thaumatin protein; WAK, wall-associated kinase.

Figure 5. Microscopic analyses of stems of healthy and HLB affected Valencia sweet orange.

A, B. Light microscopy of cross-sections of healthy young stems showing the phloem, cambium and xylem cells. F-Phloem fibers. C,D Cross section of HLB affected young stems showing greater thickness of the phloem layer compared to the healthy. Arrows point to thickened cell walls.

Figure 6. Microscopic analyses of stems of healthy and HLB affected Valencia sweet orange.

A, B, C are electron microscopy of cross- sections of healthy young stems. Arrows in A point to normal middle lamella found in healthy stem phloem. B is a lower magnification of healthy phloem showing normal companion cells (CC) on either side of sieve elements (SE) which are surrounded by phloem parenchyma cells (PP). C is an enlargement of the sieve element (SE) area pictured in B. D, E, F are cross-sections of HLB affected young stems. Arrows in D point to swollen middle lamella comparable to dark blue stained walls shown in Fig. 5. C, D. E shows phloem in affected stem comparable in magnification to B showing collapsed sieve element (CSE) surrounded by normal looking phloem parenchyma (PP) some of which contain starch (S). In upper right of micrograph is area showing swollen middle lamella (ML). F is an enlargement of area in lower left corner of E, showing collapsed sieve element (CSE) and parenchyma cells containing starch (S).

Figure 7. Microscopic analyses of roots of healthy and HLB affected Valencia sweet orange on citrumelo rootstock (Citrus paradisi x Poncirus trifoliata).

A, B. Light microscopy of cross-sections of healthy fibrous roots showing secondary thickening. The cortex and some phloem parenchyma cells are shown containing starch (S). The cortex, phloem, cambium and xylem areas are delineated by lines. C, D are cross-sections of HLB affected roots. Note that the phloem layers of these roots are comparable in thickness to those of healthy above. The arrows point to collapse, thickened cell walls.

Figure 8. Microscopic analyses of roots of healthy and HLB affected Valencia sweet orange on citrumelo rootstock (Citrus paradisi x Poncirus trifoliata).

A–D. Electron microscopy of healthy fibrous roots. A & C are relative low magnifications showing size and shape of normal phloem parenchyma (PP), sieve elements (SE) and companion cells (CC). B shows a higher magnification of same areas enhancing the sieve element areas. D–F are HLB affected roots. D is a low magnification comparable to A & C above showing the enriched cytoplasmic contents of these cells compared to the healthy above. E shows enlarged middle lamellas (ML). F shows the collapsed sieve elements (CSE). N, prominent nucleus and V, vacuole.