Homologues of CsLOB1 in citrus function as disease susceptibility genes in citrus canker

Abstract

The lateral organ boundary domain (LBD) genes encode a group of plant-specific proteins that function as transcription factors in the regulation of plant growth and development. Citrus sinensis lateral organ boundary 1 (CsLOB1) is a member of the LBD family and functions as a disease susceptibility gene in citrus bacterial canker (CBC). Thirty-four LBD members have been identified from the Citrus sinensis genome. We assessed the potential for additional members of LBD genes in citrus to function as surrogates for CsLOB1 in CBC, and compared host gene expression on induction of different LBD genes. Using custom-designed transcription activator-like (TAL) effectors, two members of the same clade as CsLOB1, named CsLOB2 and CsLOB3, were found to be capable of functioning similarly to CsLOB1 in CBC. RNA sequencing and quantitative reverse transcription-polymerase chain reaction analyses revealed a set of cell wall metabolic genes that are associated with CsLOB1, CsLOB2 and CsLOB3 expression and may represent downstream genes involved in CBC.

Keywords: citrus bacterial canker; lateral organ boundary domain (LBD) genes; susceptibility (S) genes.

Figure 1

Phylogenetic tree of Citrus sinensis lateral organ boundary domain (LBD) genes. Values with a cutoff of 70 for the bootstrap support (500 replicates) are shown on the shoulders of the branches. The scale bar indicates substitutions/site, and vertical lines are used to represent different clades of C. sinensis LBD proteins.

Figure 2

The targeted lateral organ boundary domain (LBD) genes are induced by the respective designed transcription activator‐like effectors (dTALes). (a) Validation of targeted gene induction by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Asterisks denote significant differences in the fold changes between the treatments relative to the negative control Xcc306ΔpthA4 at P < 0.05 using a t‐test. (b) CsLOB1 is not induced by the dTALes targeting CsLOB2 or CsLOB3. Different letters indicate significant differences in fold changes among the treatments at P < 0.05 using analysis of variance (ANOVA). qRT‐PCR was conducted on mRNA extracted from grapefruit leaves inoculated with the individual Xanthomonas citri ssp. citri (Xcc) strains as indicated using LBD gene‐specific primers at 5 days post‐inoculation (dpi). Expression values were normalized to the housekeeping gene orange1.1g001725m. Error bars represent the standard deviations for three independent experimental replicates. Samples were taken at 5 dpi.

Figure 3

Disease symptoms induced by designed transcription activator‐like effectors (dTALes) targeting the three CsLOB1‐related genes. (a) Light photography at 1×; (b) 6.4× magnification. Panels and inoculation sites: 1, Xcc306WT; 2, Xcc306ΔpthA4::dCsLOB2; 3, Xcc306ΔpthA4::dCsLOB3; 4, Xcc306ΔpthA4::dCsLOB4; 5, Xcc306ΔpthA4; 6, healthy leaf. Photographs were taken at 15 days post‐inoculation (dpi) of various strains as shown in the figures. Inocula were 10³ times dilutions of bacterial suspensions with an optical density at 600 nm (OD₆₀₀) of 0.5.

Figure 4

dCsLOB2 promoted an increase in the bacterial population in host plants. Bacterial populations were measured for three strains (Xcc306ΔpthA4, Xcc306WT and Xcc306ΔpthA4::dCsLOB2) on grapefruit. The x‐axis shows the days post‐inoculation. Asterisks indicate significant difference (P < 0.01) of two comparisons: Xcc306WT and Xcc306ΔpthA4::dCsLOB2 vs. Xcc306ΔpthA4. Statistical significance of the analysis was tested using t‐test. cfu, colony‐forming unit.

Figure 5

S gene expression does not contribute to enhanced virulence of a non‐host pathogen. (a) Pustule‐like symptoms were observed when DCsLOB2 was transformed into Xanthomonas euvesicatoria Xcv85‐10. Photographs were taken at 30 days post‐inoculation (dpi) of grapefruit leaves with Xcv85‐10::pHM1 or Xcv85‐10::dCsLOB2 at 1× (1–4) or 6.4× (5, 6) magnification. Inoculum with an optical density (OD₆₀₀) of 0.5 (1,2) or the same inoculum diluted 10³ times (3–6). (b) Bacterial population assay of the two strains Xcv85‐10::pHM1 and Xcv85‐10::dCSLOB2 on grapefruit. t‐test (P < 0.05) showed no significant difference between the two treatments. cfu, colony‐forming unit.

Figure 6

RNA‐sequencing (RNA‐Seq) results of the gene expression profile induced by CsLOB1, CsLOB2, CsLOB3 or CsLOB4. Venn diagrams show the number of genes that were significantly differentially expressed in each comparison (List1, Xcc306WT vs. 306ΔpthA4; List2, 306ΔpthA4::dCsLOB2 vs. 306ΔpthA4; List3, 306ΔpthA4::dCsLOB3 vs. 306ΔpthA4; List4, 306ΔpthA4::dCsLOB4 vs. 306ΔpthA4). The comparisons are represented by blue, yellow, green or red ovals. Genes that show significant differences in more than one comparison are plotted in the overlapping areas. The black numbers denote the total significantly regulated genes at a false discovery rate (FDR) < 0.05, the red numbers denote the up‐regulated genes and the yellow numbers denote the down‐regulated genes.

Figure 7

Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) validation of cell wall‐related genes induced by the expression of CsLOB1, CsLOB2, CsLOB3 and CsLOB4. RNA samples were grapefruit leaves treated with various Xanthomonas citri ssp. citri (Xcc) strains as indicated in the figure, and were taken at 5 days post‐inoculation (dpi). Inoculum had an optical density at 600 nm (OD₆₀₀) of 0.5. Expression values were normalized to the housekeeping gene orange1.1g001725m. Error bars represent the standard error for three independent experimental replicates. Different letters indicate significant differences among the various treatments for each test gene at the significance level of P < 0.05 according to analysis of variance (ANOVA). Different genes are represented by abbreviations of the annotations of various transcripts. Gene IDs are from Phytozome. EβG, endo β‐1,4‐glucanase (orange1.1g009690m); GH5, glycosyl hydrolase family 5 (orange1.1g014426m); PG, polygalacturonase (orange1.1g043061m); PL1, pectate lyase1 (orange1.1g015623m); PL2, pectate lyase2 (orange1.1g011814m); αE, α‐expansin (orange1.1g024916m); βE1, β‐expansin1 (orange1.1g023962m); βE2, β‐expansin2 (orange1.1g024303m).

Figure 8

Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) validation of genes up‐regulated by the expression of CsLOB4, which was induced by either dCsLOB4 or dCsLOB4‐2. RNA samples were grapefruit leaves treated with various Xanthomonas citri ssp. citri (Xcc) strains as indicated in the figure, and were taken at 5 days post‐inoculation (dpi). Inoculum had an optical density at 600 nm (OD₆₀₀) of 0.5. Expression values were normalized to the housekeeping gene orange1.1g001725m. Different genes are represented by the transcript ID from Phytozome. Error bars represent the standard error for three independent experimental replicates. Single asterisks indicate a significant difference relative to 306ΔpthA4 at the significance level of P < 0.05 according to t‐test.