Different transcriptional response to Xanthomonas citri subsp. citri between kumquat and sweet orange with contrasting canker tolerance

Abstract

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating biotic stresses affecting the citrus industry. Meiwa kumquat (Fortunella crassifolia) is canker-resistant, while Newhall navel orange (Citrus sinensis Osbeck) is canker-sensitive. To understand the molecular mechanisms underlying the differences in responses to Xcc, transcriptomic profiles of these two genotypes following Xcc attack were compared by using the Affymetrix citrus genome GeneChip. A total of 794 and 1324 differentially expressed genes (DEGs) were identified as canker-responsive genes in Meiwa and Newhall, respectively. Of these, 230 genes were expressed in common between both genotypes, while 564 and 1094 genes were only significantly expressed in either Meiwa or Newhall. Gene ontology (GO) annotation and Singular Enrichment Analysis (SEA) of the DEGs showed that genes related to the cell wall and polysaccharide metabolism were induced for basic defense in both Meiwa and Newhall, such as chitinase, glucanase and thaumatin-like protein. Moreover, apart from inducing basic defense, Meiwa showed specially upregulated expression of several genes involved in the response to biotic stimulus, defense response, and cation binding as comparing with Newhall. And in Newhall, abundant photosynthesis-related genes were significantly down-regulated, which may be in order to ensure the basic defense. This study revealed different molecular responses to canker disease in Meiwa and Newhall, affording insight into the response to canker and providing valuable information for the identification of potential genes for engineering canker tolerance in the future.

Figure 1. Comparison of canker disease development in ‘Meiwa’ and ‘Newhall’.

(A) Leaves of ‘Meiwa’ and ‘Newhall’ were pinprick-inoculated with citrus canker bacterium and periodically observed within 7 d, and the pictures were taken at 5 and 7 days post inoculation (DPI). (B) Quantitative comparison of the bacterial population at the inoculation sites of ‘Meiwa’ and ‘Newhall’ leaves after 6 days inoculation.

Figure 2. Number of differentially expressed genes in ‘Meiwa’ and ‘Newhall’ after statistical analysis.

(A) Number of significantly upregulated and downregulated genes in ‘Meiwa’ and ‘Newhall’. (B–C) Venn diagram shows the number of upregulated (B) and downregulated (C) genes that are expressed in common or in special between ‘Meiwa’ and ‘Newhall’.

Figure 3. Verification of the microarray results by semi-quantitative RT-PCR.

(A) RT-PCR results of the genes. The cDNA of ‘Meiwa’ and ‘Newhall’ leaves sampled at 0 (designated as M0 and N0, respectively) and 5 (designated as M5 and N5, respectively) days post inoculation (DPI) was amplified with specific primers of the selected genes, using Actin as a control. (B) Comparison of the expression ratios (M5/M0 and N5/N0) between RT-PCR analysis and the microarray data. The expression ratios were calculated by quantifying the band density using the Quantity One software.

Figure 4. Functional categorization of the common upregulated and downregulated genes in ‘Meiwa’ and ‘Newhall’ based on the GO annotation.

Figure 5. Functional categorization of 380 upregulated and 184 downregulated genes that are specifically regulated in ‘Meiwa’ based on GO annotation.