Transcriptome Profiling of Citrus Fruit Response to Huanglongbing Disease

Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production

HLB-altered expression of hormone and transcription factors.

<p>Differential expression of (A) hormone-related transcripts and (B) transcription factors. Color scale indicates the comparisons between symptomatic asymptomatic and apparently healthy fruit, same as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038039#pone-0038039-g003" target="_blank">Figure 3</a>.</p

List of genes analyzed by quantitative RT-PCR, including P-values for each of three pairwise comparisons from ANOVA of the expression data.

<p>Names of genes (or abbreviated gene names in parentheses) correspond to bar clusters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038039#pone-0038039-g007" target="_blank">Figure 7</a>. Symptomatic (SY), asymptomatic (AS), apparently healthy (AH), healthy control (CO). P-values<0.05 are considered significant.</p

MapMan views of differentially regulated primary metabolism genes.

<p>Symptomatic fruit was compared to asymptomatic fruit within the same tree (AS), different tree (AH) and both trees. A) Light reactions of photosynthesis. B) Glycolysis. “CB” block shows gene expression in cytosolic branch genes of small carbohydrate metabolism. C) Sucrose and starch metabolism D) Raffinose metabolism. Colored data points indicate up or down expression with log fold ratios >0.5 and <−0.5, respectively.</p

Differentially regulated pathways (up- and down-regulated) involved in HLB response in the symptomatic fruit as determined using Pathexpress.

<p>Analysis was performed on the list of genes differentially expressed at a significance level of p<0.1 comparing symptomatic to asymptomatic fruits within tree and between different trees “N.s.” means not significant.</p

Fruit metabolism and regulatory pathways in CaLas-infected fruits.

<p>Genes, pathways, and cell functions that were differentially expressed are indicated with a square (red for up-regulated, green for down-regulated). Significantly differentially regulated pathways in gene set enrichment analysis are indicated in yellow.</p

HLB-altered expression related to protein degradation and misfolding.

<p>A) Differentially expressed genes involved in ubiquitin-dependent degradation, showing comparison between symptomatic and apparently healthy fruits. B) Pattern of expression of heat shock protein 82 (HSP82) in HLB-free location (CO), apparently healthy (AH), asymptomatic (AS), and symptomatic (SY) fruits. C) Protein-protein interaction network predicted in <i>Citrus</i> based on the <i>Arabidopsis</i> knowledgebase. Networks between proteins encoded by HLB-regulated genes were divided into four different clusters. Node legend indicates functional classes of transcripts.</p

HLB-altered expression in symptomatic fruit in volatile and defense response pathways.

<p>Log fold ratios are indicated by a gradient from red (up-regulated) to green (down-regulated). Comparison was made between symptomatic and apparently healthy fruits from the infected orchard. A) Jasmonate biosynthesis, B) Terpenoid and carotenoid biosynthesis, C) Salicylic acid pathway.</p

qRT-PCR analysis.

<p>Pattern of expression of genes belonging to different pathways in the four categories of fruits: symptomatic (SY), asymptomatic (AS), apparently healthy (AH) and healthy from HLB-free location (CO). Gene names correspond to those listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038039#pone-0038039-t002" target="_blank">Table 2</a>. Bar heights for expression of Invertase also shown at 20-fold magnification (lower-right inset).</p

Functional categorization of differentially regulated genes in symptomatic fruits.

<p>Metabolism overview in MapMan depicting differential gene expression in symptomatic and apparently healthy fruits from the infected orchard. Log fold ratios are indicated as a gradient between red (up-regulated) and green (down-regulated).</p