Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype

Abstract

Background: Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam.

Results: The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas.

Conclusions: Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.

Figure 1

Distribution of differentially expressed genes (DEGs) (x-axis) into Gene Ontology (GO) categories (biological process) (y-axis) according to Gene Set Enrichment Analysis (GSEA). Only biological processes (BPs) discussed in the results are presented here. A complete list of BPs can be found in Additional file 2.

Figure 2

Average expression stability values (M) and Pairwise variation (V) of the ten citrus reference genes calculated by geNorm. A) A lower M value indicates more stable expression. B) Pairwise variation (V) to determine the optimal number of reference genes suggested by geNorm to a reliable normalization.

Figure 3

Comparison of the expression levels of a subset of ten genes in symptomatic (SY) and asymptomatic (AS) leaves of Hamlin sweet orange infected with CaLas or CaLam in relation to their controls (H) by RT-qPCR. Comparisons were performed by a nonparametric one-way ANOVA with 1000 unrestricted permutations, followed by pair-wise comparisons with Bonferroni adjustment. Levels of significance less than or equal to 0.05 and 0.1 were considered as “significant” (*) and “suggestive” (^.), respectively. The remaining ten genes tested by RT-qPCR can be found in Additional file 5.