A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall-degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N- and C-terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild-type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence-promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.

Keywords: Xanthomonas oryzae pv. oryzae; cellulases; endoglucanase; exoglucanase; rice.

Figure 1

Crystal structure of the catalytic domain of CbsA. (A) CbsA has a central distorted β‐barrel (pink) made up of eight strands. The N‐ and C‐terminal loops are shown in blue and red, respectively, whereas the unique loops present towards the C‐terminal region are shown in cyan. (B) The CbsA structure with the modelled substrate. The surface representation of the enzyme represents a well‐defined substrate‐binding tunnel responsible for processive hydrolysis of the single‐chain cellulose polymer. Three molecules of cellobiose were modelled from PDB ID: 4B4F.

Figure 2

Multiple sequence alignment of the CbsA catalytic domain and putative catalytic residues. (A) Multiple sequence alignment of the CbsA catalytic domain with homologous regions from other bacteria and fungi. Residues marked with a green triangle are conserved tryptophans lining the tunnel and blue triangles indicate the putative catalytic residues. The sequences of the bacterial‐specific loops are enclosed in green boxes. (B) Putative catalytic residues present in close proximity to the modelled substrate. The subsites of the substrate‐binding tunnel are numbered from the non‐reducing end to the reducing end as −2, −1, +1, +2, +3 and +4. Residues around the active site are shown with sticks.

Figure 3

D131A CbsA displays an endo mode of activity. (A) Equal amounts of purified preparations of wild‐type (Wt) CbsA, D131A CbsA and ClsA proteins were added to wells in carboxymethylcellulose (CMC) plates and stained with Congo red after 36–48 h of incubation, as described in Experimental procedures. The presence of a halo around the well indicates the activity of the enzyme on CMC. (B) Separation of hydrolysed products by thin layer chromatography (TLC). The CMC substrate was incubated with buffer, Wt CbsA, D131A CbsA and ClsA, and the complete reaction mixture was spotted on a TLC sheet in the following lanes: 1, CMC + buffer; 2, CMC + Wt CbsA; 3, CMC + ClsA; 4, CMC + D131A CbsA. A mix of the oligosaccharides glucose to cellohexaose (G–G6) was loaded as a ladder in the lane labelled as M. Sugars present in the reaction mixture were separated using butanol, acetic acid and water in a 2 : 1 : 1 ratio as the mobile phase, and were detected using orcinol and sulfuric acid reagent, as described in Experimental procedures. (C) Viscometric analysis of CMC solution on activity of Wt CbsA, D131A CbsA and ClsA. The flow time of the reaction mixture containing CMC together with buffer, Wt CbsA, D131A CbsA and ClsA was measured at regular intervals using a viscosity bath maintained at a constant temperature of 37 ^οC. As described in Experimental procedures, the kinematic viscosity of the samples was calculated and plotted against the incubation time. (D) Activity of Wt CbsA, D131A CbsA and ClsA on soluble oligosaccharides. The oligosaccharide substrates cellobiose G2 (1), cellotriose G3 (2), cellotetraose G4 (3), cellopentaose G5 (4) and cellohexaose G6 (5) were incubated with Wt CbsA, D131A CbsA and ClsA, and the complete reaction mixture was loaded onto the TLC sheet. The sugars released were separated and detected as described in Experimental procedures. A mix of oligosaccharides (G–G6) is shown in the lane labelled as M.

Figure 4

Virulence phenotype of D131A mutant CbsA. (A) Rice leaves were inoculated with wild‐type (Wt) Xanthomonas oryzae pv. oryzae (Xoo) (BXO43) (1), the cbsA^– mutant (2), the cbsA^– mutant + pHM1 (3), the cbsA^– mutant + pHM1::Wt cbsA (4) and the cbsA^– mutant + pHM1::D131A cbsA (5). At 20 days post‐inoculation, the images of infected rice leaves were captured. (B) Lesion lengths were measured after 20 days. Error bars indicate the standard deviation of readings from at least 10 inoculated leaves. Similar results were obtained in independent experiments. A Student's two‐tailed t‐test for independent means was performed for the following groups: wild‐type and cbsA mutant, mutant with empty vector (control strain), mutant expressing Wt CbsA (complemented strain) and mutant expressing D131A CbsA. *All the compared values were significantly different at the P < 0.05 level.

Figure 5

D131A mutation does not affect the ability of CbsA to induce defence responses in rice tissues. (A) Callose deposition in rice leaves: 10–15‐day‐old rice leaves were infiltrated with 100 µL of buffer, Wt CbsA or D131A CbsA proteins of concentration 0.1 mg/mL. The leaves were subsequently treated to remove chlorophyll, stained with aniline blue and visualized under an epifluorescence microscope. Bright spots in the images are the callose deposits. (B) Programmed cell death in rice roots: roots of 2–3‐day‐old rice seedlings were excised and treated with buffer, Wt CbsA or D131A CbsA proteins (0.5 mg/mL) for 16 h, stained with propidium iodide (PI) and examined under a confocal microscope. Extensive internalization of PI and dispersal within the cell are indicative of programmed cell death (PCD). Scale bar measures 20 µm. The concentrations of protein used are the lowest effective concentrations for the wild type protein to elicit callose deposition and programmed cell death respectively.

Figure 6

D131A CbsA is as efficient as the wild‐type (Wt) protein in the induction of callose deposition in rice leaves in vivo. (A) Ten‐ to 15‐day‐old rice leaves were infiltrated with one of the following strains of Xanthomonas oryzae pv. oryzae: T3SS^– mutant, T3SS^– cbsA ^– double mutant, T3SS^– cbsA ^– + pHM1 (empty vector control), T3SS^– cbsA ^– + pHM1::cbsA (Wt complemented clone), T3SS^– cbsA ^– + pHM1::D131AcbsA and BXO43 (Wt). The leaves were subsequently treated to remove chlorophyll, stained with aniline blue and visualized under an epifluorescence microscope. Bright spots in the images are the callose deposits. (B) The average numbers of callose spots from at least four leaves and three to four different viewing areas in each experiment were plotted. Error bars represent standard deviation (SD). A Student's two‐tailed t‐test for independent means was performed in pairwise combinations for all the values. Values with the same letter (either a or b) are not significantly different at P < 0.05. Similar results were obtained in independent experiments.