Fructose-bisphophate aldolase exhibits functional roles between carbon metabolism and the hrp system in rice pathogen Xanthomonas oryzae pv. oryzicola

Abstract

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N(9)-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.

Figure 1. fbaB is required for full virulence and growth of X. oryzae pv. oryzicola in planta.

(A) Symptoms caused by different X. oryzae pv. oryzicola strains suspended in water to OD₆₀₀ = 0.3 (approximately 1×10⁸ cfu/ml) on inoculated leaves of the host rice cv. Shanyou63 (susceptible cultivar) (2-month-old) by leaf-needling inoculation. Photographs were taken 14 days post-inoculation. RS105, the wild-type strain; RΔfbaB, the fbaB deletion mutant; CRΔfbaB, the complemented strain of RΔfbaB with the fbaB gene; RΔhrcV, a type III-deficient strain as a negative control. (B) Lesion lengths of rice bacterial leaf streak caused by different X. oryzae pv. oryzicola strains in rice. Values are the means ± standard deviations (SD) from three repeats, each with five leaves. The different symbol in each horizontal data column results from a paired, two-tailed Student t test relative to the wild-type. **, P = 0.01. (C) Bactrial growth capacity in inoculated leaves. Bacteria were recovered from the inoculated leaves every 24 hours in a period of 4 days post inoculation, and homogenized in sterile water. The homogenates were diluted and plated on NA plates with appropriate antibiotics. Bacterial CFU were counted after incubation at 28°C for 3 days. Data are the mean ± SD from three repeats.

Figure 2. Growth curves of X. oryzae pv. oryzicola in sole carbon media.

RS105, the wild-type strain; RΔfbaB, the fbaB deletion mutant; CRΔfbaB, the complemented strain of RΔfbaB with the fbaB gene. The initial concentration of the tested strains was adjusted to OD₆₀₀ of 0.1 with NCM supplemented with fructose, pyruvate or malate as the sole carbon source. Aliquots were taken in triplicate at intervals of 120 h after incubation at 28°C, and bacterial growth was determined by measuring OD₆₀₀ against the medium blank. Values given are the means ± SD of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments.

Figure 3. The expression of fabB in X. oryzae pv. oryzicola is negatively by hrpX and hrpG in rice suspension cells.

(A) Schematic map of the promoter region containing PIP-box and -10 box-like motif of fabB fused with a promoterless gusA gene. * stands for base substitutions. The constructs are listed on the right. (B) Expression analysis of fbaB by real-time quantitative RT-PCR. RNAs were isolated from cultures of the wild-type RS105, the hrpG deletion mutant RΔhrpG and the hrpX deletion mutant RΔhrpX strains which were grown in NB medium and rice suspension cells for 16 h, respectively. The relative mRNAs level of fbaB was calculated with respect to the level of the corresponding transcript in the wild-type RS105. (C) Effects of the mutated PIP-box on gusA transcript. The gusA transcript level by the wild-type PIP-box promoter (a) and three base-substituted PIP-box promoter (b, c, d) in the wild-type RS105, the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpX were investigated, respectively. All the reporter strains above were cultured in rice suspension cells for 16 h and gusA transcript levels were then determined by real-time PCR. The transcript of gusA in the wild-type was taken as one unit. Data are the mean ± SD of triplicate measurements from a representative experiment; and similar results were obtained in two other independent experiments. The asterisks in each horizontal data column indicate significant differences at P = 0.01 by t test.

Figure 4. Effects of different carbohydrates on the expression of hrpX, hrpG and fbaB in X. oryzae pv. oryzicola.

RNAs were isolated from cultures of the wild-type RS105 strain and the fbaB deletion mutant RΔfbaB grown in NY medium alone and NY supplemented with 0.5% of various carbohydrates for 16 h. The relative mRNAs levels of hrpX, hrpG and fbaB genes were calculated by real-time quantitative RT-PCR with respect to the level of the corresponding transcript in the wild-type RS105 cultured in NY medium alone. Data presented are the means ± SD of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments.

Figure 5. Influence of the mutation in fbaB on the expression of hrp-hrc-hpa genes of X. oryzae pv. oryzicola.

(A) Semi-quantitative RT-PCR analysis. RNAs were isolated from cultures of the wild-type RS105 strain and the fbaB mutant RΔfbaB grown in rice suspension cells for 16 h. The 16S rRNA gene of the pathogen is used as the standard internal control. The tested hrp-hrc-hpa genes were selected based on the reports , , with the primer pairs (Table S1) and the sequence of the hrp clusters (AF272885, AY875714) was used as the reference. (B) Real-time quantitative RT-PCR analysis. The relative mRNA level of the tested hrp-hrc-hpa genes in the fbaB mutant RΔfbaB was calculated with respect to the level of the corresponding transcripts in the wild-type RS105 cultured in rice suspension cells for 16 h. Values given are the means ± SD of triplicate measurements from a representative experiment. The asterisks in each horizontal data column indicate significant differences. **, P = 0.01, t test. Experiment was repeated twice and yielded similar results.

Figure 6. The mutation in hrcC, hrpE and hpa3 reduced the ability of X. oryzae pv. oryzicola to acquire pyruvate and malate.

RS105, the wild-type strain; RΔhrcC, the hrcC deletion mutant; RΔhrpE, the hrpE deletion mutant; RΔhpa3, the hpa3 deletion mutant. The initial concentration of the tested strains was adjusted to OD₆₀₀ of 0.05 with NCM supplemented with pyruvate or malate as the sole carbon source. Aliquots were taken in triplicate at intervals of 120 h after incubation at 28°C, and bacterial growth was determined by measuring OD₆₀₀ against the medium blank. Values given are the means ± SD of triplicate measurements from a representative result of other two similar independent experiments.

Figure 7. Working model of FbaB coordinating with hrp genes of X. oryzae pv. oryzicola in carbon metabolic pathoways.

The lined arrows from the carbohydrates in dashed-line boxes or the double lined arrows from the intermediates indicate carbon flows in glycolysis, gluconeogenesis, pentose phosphate pathway (PPP), entner–doudoroff (ED) and tricarboxylic acid (TCA) cycle pathways, respectively. The grey box displays fbaB encodes a fructose-bisphophate aldolase that converts vertically fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The mutation in fbaB has no influence on ED and PPP pathways, but impairs glycolysis of the pathogen to use fructose and block gluconeogenesis to use pyruvate and malate. The expression of an unknown regulator in a cycled question mark may be enhanced by galactose, glucose, mannose, sucrose, fructose and pyruvate (as shown by dash-lined arrow) and repressed by malate (a dash-lined arrow with a stop bar). The unkown factor may differentially regulate the expression of hrpG or/and hrpX which down-regulate the expression of fbaB (a dash-lined arrow with a stop bar). The unknown regulator may also control the transcripts of hrcC, hrpE and hpa3 (other than other hrp-hrc-hpa genes) which are not completely regulated by HrpG and HrpX . Being the components of the T3SS apparatus, HrcC, HrpE and Hpa3 may faciliate X. oryzae pv. oryzicola to utilize the intermediates, like pyruvate and malate, of the TCA cycle from plants.