Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria

Abstract

The avrBs2 avirulence gene of the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria triggers disease resistance in pepper plants containing the Bs2 resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper Bs2 gene on the evolution of avrBs2 by characterizing the molecular basis for virulence of 20 X. campestris pv. vesicatoria field strains that were isolated from disease spots on previously resistant Bs2 pepper plants. All field strains tested were complemented by a wild-type copy of avrBs2 in their ability to trigger disease resistance on Bs2 plants. DNA sequencing revealed four mutant alleles of avrBs2, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of avrBs2. The other two avrBs2 alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic X. campestris pv. vesicatoria strains by chromosomal avrBs2 gene exchange to study the effects of these mutations on the dual functions of avrBs2 in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of avrBs2-induced resistance on Bs2 pepper plants and abolition of avrBs2-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of avrBs2 on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on Bs2 plants. Consistent with the divergent selection pressures on avrBs2 exerted by the Bs2 resistance gene, our results show that avrBs2 is evolving to decrease detection by the Bs2 gene while at the same time maintaining its virulence function.

FIG. 1

Schematic representation of AvrBs2 and positions of mutations. The region of homology with the phosphodiester synthase ACS is hatched. Nucleotide changes in avrBs2-1 and avrBs2-2 are shown above the scheme, with numbering denoting the nucleotide positions of avrBs2⁺ (34). Single amino acid (aa) changes generated by avrBs2-3 and avrBs2-4 are shown below, with numbering denoting amino acid positions and with the assumption of the start of transcription being at the codon of nucleotides 179 to 181 (34). The alignment between ACS and AvrBs2 (residues 173 to 714) was performed using the BLAST algorithm (1), and only the region surrounding the single amino acid changes is shown.

FIG. 2

Protein gel blot analysis of X. campestris pv. vesicatoria crude lysate. The locations of the 113- and 54-kDa molecular mass markers are shown. The indicated avrBs2 alleles on the vector pDD62 were conjugated into strain GM98-38. Approximately 30 μg of total protein was loaded onto a 12% polyacrylamide gel. AvrBs2 was detected using polyclonal antibodies raised against full-length AvrBs2. The arrow shows the position of full-length AvrBs2 at approximately 80 kDa. The arrowhead indicates a nonspecific protein recognized by the AvrBs2 antiserum.

FIG. 3

In planta growth of X. campestris pv. vesicatoria (Xcv) strains GM98-38 (A) and GM97-1 (B) in the pepper cultivars ECW-0 (bs2/bs2, open symbols) and ECW-20R (Bs2/Bs2, filled symbols). The growth of original field strains (squares) and isogenic strains expressing avrBs2⁺ (circles) is shown. Values are means of results with triplicate samples. Error bars denote standard deviation and are shown where values were larger than those represented by the symbols. Similar results were obtained in two independent experiments.

FIG. 4

In planta growth in ECW-0 (A) and ECW-20R (B) of X. campestris pv. vesicatoria strain GM98-38 expressing avrBs2⁺ (filled squares) and the mutant alleles avrBs2-2 (filled diamonds), avrBs2-3 (open circles), and avrBs2-4 (open triangles). Values are means of results with triplicate samples. Error bars denote standard deviation and are shown where values were larger than the values represented by the symbols. Similar results were obtained in three independent experiments. (C) Data from day 8 postinoculation in panels A and B in bar graph format for ease of comparison of levels of bacterial growth in ECW-0 (open bars) and ECW-20R (filled bars) pepper plants. Error bars denote standard deviation and are shown where values were larger than those represented by the bar outlines. del, deletion.

FIG. 5

Phenotype in leaves of ECW-0 (A) and ECW-20R (B) inoculated with 10⁹ CFU of X. campestris pv. vesicatoria strain GM98-38 expressing avrBs2⁺ (upper right) and the mutant alleles avrBs2-2 (upper left), avrBs2-3 (lower left), and avrBs2-4 (lower right) per ml. Phenotypes were recorded 3 days postinoculation by placing leaves on a light box to distinguish clear water-soaked lesions (disease) from browning of the tissue associated with HR (resistance). del, deletion.

FIG. 6

Pulsed-field gel electrophoresis of genomic DNA isolated from the indicated X. campestris pv. vesicatoria (Xcv) field strains digested with SpeI. The locations of the 680-, 365-, and 225-kb markers are shown. Arrows indicate polymorphisms that distinguish strain GM98-38 from the other strains analyzed.