A eukaryotic-acquired gene by a biotrophic phytopathogen allows prolonged survival on the host by counteracting the shut-down of plant photosynthesis

Abstract

Xanthomonas citri pv. citri, the bacteria responsible for citrus canker posses a biological active plant natriuretic peptide (PNP)-like protein, not present in any other bacteria. PNPs are a class of extracellular, systemically mobile peptides that elicit a number of plant responses important in homeostasis and growth. Previously, we showed that a Xanthomonas citri pv. citri mutant lacking the PNP-like protein XacPNP produced more necrotic lesions in citrus leaves than wild type infections and suggested a role for XacPNP in the regulation of host homeostasis. Here we have analyzed the proteome modifications observed in citrus leaves infected with the wild type and XacPNP deletion mutant bacteria. While both of them cause down-regulation of enzymes related to photosynthesis as well as chloroplastic ribosomal proteins, proteins related to defense responses are up-regulated. However, leaves infiltrated with the XacPNP deletion mutant show a more pronounced decrease in photosynthetic proteins while no reduction in defense related proteins as compared to the wild-type pathogen. This suggests that XacPNP serves the pathogen to maintain host photosynthetic efficiency during pathogenesis. The results from the proteomics analyses are consistent with our chlorophyll fluorescence data and transcript analyses of defense genes that show a more marked reduction in photosynthesis in the mutant but no difference in the induction of genes diagnostic for biotic-stress responses. We therefore conclude that XacPNP counteracts the shut-down of host photosynthesis during infection and in that way maintains the tissue in better conditions, suggesting that the pathogen has adapted a host gene to modify its natural host and render it a better reservoir for prolonged bacterial survival and thus for further colonization.

Figure 1. Protein profiles in 2-DE of total soluble proteins of Xcc-infected citrus leaves.

Equal amounts of urea-buffer extracted proteins (200 µg) were separated on 7 cm pI 4–7 linear gradient strips in the first dimension and on 12% SDS-PAGE in the second dimension and stained with Coomassie blue. Proteins with significantly different expression levels between control and infected plants were marked with circles and numbered. Numbers refer to protein spot numbers on Table 1. Numbers on the right indicate molecular mass in kilodalton (kDa). (A) Citrus leaves infiltrated with Tris solution as control. (B) Citrus leaves inoculated with X. citri pv. citri 3 dpi and (C) 6 dpi.

Figure 2. Protein profiles in 2-DE of total soluble proteins of Xcc and ΔXacPNP-infected citrus leaves.

Equal amounts of urea-buffer extracted proteins (200 µg) were separated on 7 cm pI 4–7 linear gradient strips in the first dimension and on 12% SDS-PAGE in the second dimension and stained with Coomassie blue. Spots numbers are the same as in Figure 1. Numbers on the right indicate molecular mass in kilodalton (kDa). (A) Citrus leaves inoculated with Xcc wild type (XccWT) 3 dpi and (C) 6 dpi and (B) with ΔXacPNP 3 dpi and (D) 6 dpi.

Figure 3. Photosynthetic parameters measurements.

(A) Potential quantum efficiency of PSII (F _v/F _m) of control (filled triangles), XccWT (open circles) and ΔXacPNP (solid circles) infiltrated citrus leaves (B) Effective quantum efficiency of PSII (F′_v/F′_m) (C) PSII operating efficiency (φ_PSII). (D) Photochemical fluorescence quenching (qP). (E) Nonphotochemical fluorescence quenching (NPQ). B–E were measured in control, XccWT and ΔXacPNP infiltrated citrus leaves as represented as in A. (F) CO₂ assimilation measured on infiltrated citrus leaves at 48 hpi. The results are the mean of five replicates and error bars represent the standard deviations.

Figure 4. Expression of defense-related genes in citrus leaves infected with ΔXacPNP and Xcc (XccWT).

RT-PCR were performed in RNA samples taken at 1, 4 and 24 hpi. MKK4, MAP kinase kinase 4; PRXa, peroxidorexin; Rboh, NADPH oxidase; GST, glutathione-S-transferase; PAL, phenylalanine ammonia lyase; HMGR, 3-hydroxy-methylglutaryl CoA reductase; Lox2, lipoxygenase 2. As controls (C) leaves infiltrated with 10 mM MgCl₂ at the times stated were analyzed. While in the figure the control at 1 hpi is shown, similar results were obtained for 4 and 24 hpi.

Figure 5. Model of XacPNP action in citrus canker.

Typical citrus leaves lesions with ΔXacPNP (left) and Xcc wild type (right) are observed in the bottom images where differences in tissue necrosis are shown. On the leaf is a proposed model for PNP-dependent cellular processes (described in the text). Solid arrows indicate experimentally confirmed functions and dashed arrows indicate proposed mechanisms.