Digital Gene Expression Analysis of Ponkan Mandarin (Citrus reticulata Blanco) in Response to Asia Citrus Psyllid-Vectored Huanglongbing Infection

Abstract

Citrus Huanglongbing (HLB), the most destructive citrus disease, can be transmitted by psyllids and diseased budwoods. Although the final symptoms of the two main HLB transmission ways were similar and hard to distinguish, the host responses might be different. In this study, the global gene changes in leaves of ponkan (Citrus reticulata) mandarin trees following psyllid-transmission of HLB were analyzed at the early symptomatic stage (13 weeks post inoculation, wpi) and late symptomatic stage (26 wpi) using digital gene expression (DGE) profiling. At 13 wpi, 2452 genes were downregulated while only 604 genes were upregulated in HLB infected ponkan leaves but no pathway enrichment was identified. Gene function analysis showed impairment in defense at the early stage of infection. At late stage of 26 wpi, however, differentially expressed genes (DEGs) involved in carbohydrate metabolism, plant defense, hormone signaling, secondary metabolism, transcription regulation were overwhelmingly upregulated, indicating that the defense reactions were eventually activated. The results indicated that HLB bacterial infection significantly influenced ponkan gene expression, and a delayed response of the host to the fast growing bacteria might be responsible for its failure in fighting against the bacteria.

Keywords: Asian citrus psyllids (ACP); Citrus Huanglongbing (HLB); differentially expressed genes (DEGs); digital gene expression (DGE); ponkan mandarin.

Figure 1

Differential regulation of genes in leaves at 13 and 26 weeks post inoculation (wpi). Figures in the two parentheses indicate total numbers of differentially expressed genes (DEGs) identified in leaves 13 and 26 wpi, respectively. Figures before virgules indicate numbers of upregulated DEGs, and those after virgules indicate the downregulated.

Figure 2

Overview of the DEGs involved in metabolisms at 13 (A) and 26 wpi (B) respectively in ponkan leaves infected with Asian citrus psyllid (ACP)-vectored CLas bacteria. The red show the genes that were significantly upregulated while the green show the significantly downregulated. CHO: Carbohydrate; TCA: Tricarboxylic acid cycle; OPP: Oxidative Pentose Phosphate; * Raffinose family metabolism; Grey dots: genes without significant expression difference, arrows were used to indicate the direction of the pathways.

Figure 3

Stress related DEGs regulated by CLas infection at 13 wpi (A) and 26 wpi (B) respectively in ponkan leaves. Genes significantly upregulated by CLas infection are displayed in red, and those downregulated are shown in green. HSPs: heat-shock proteins; bZIP: basic leucin zipper; ERF: Ethylene response factor; MAPK: mitogen-activated protein kinase; MYB: MYB family transcription factor; DOF: DNA binding with one finger. ABA: abscisic acid; SA: salicylic acid; JA: jasmonic acid; R gene: resistance gene; Misc.: miscellaneous, PR-protein: pathogenesis-related protein, WRKY: WRKY transcription factors, DOF: DNA-binding with one finger.

Figure 4

MapMan views of transport related genes that were differentially regulated after CLas infection at 13 (A) and 26 wpi (B). Genes significantly upregulated by CLas are displayed in red, and the downregulated are shown in green. * PIP (plasma membrane intrinsic protein) genes; SIP: small and basic intrinsic protein genes; NIP: Nod26-like intrinsic protein genes; TIP: tonoplast intrinsic protein genes; grid framed with ellipse represents chloroplast.

Figure 5

Comparison of the expression levels in ponkan leaves infected with CLas relative to their controls of six of the representative genes obtained by real-time PCR (A,C) and by digital gene expression (DGE) profiles (B,D). A,B: at 13 wpi; C,D: at 26 wpi. * and ** indicate significant changes at p < 0.05 and p < 0.01, respectively. MYB, MYB family transcription factor; HSP, heat-shock proteins; KAR, ketol-acid reductoisomerase; BG, β-1,3-glucanase; ERF, Ethylene response factor.