A novel class of gene controlling virulence in plant pathogenic ascomycete fungi

Abstract

Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence. One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype. Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains. CPS1 orthologs were detected in all Cochliobolus spp. examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants. Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketidepeptide hybrids. Disruption of CPS1 caused reduced virulence of both race T and race O of C. heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat. These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes.

Figure 1

REMI mutant R.C4.2696 has reduced virulence on corn. (A) Virulence test on N-cytoplasm corn. The mutant R.C4.2696 (Right) produced smaller lesions (compare those at arrows) than WT strain C4 (Left). (B) T-cytoplasm corn inoculated with (Left to Right) mutant R.C4.2696 (Tox1⁺;cps1⁻), WT race T (strain C4, Tox1⁺;CPS1⁺), and a Tox1⁺;cps1⁻ progeny (1301-R-26); symptoms (three leaves per strain) recorded at 5 days. Leaves infected by WT collapsed (arrow); those infected by Tox1⁺;cps1⁻ mutants remained alive despite sustaining small lesions. (C) Microbial assay for T-toxin production. Plate containing T-toxin-sensitive Escherichia coli cells was inoculated with agar blocks bearing mycelia of the three strains shown in B (same order); (Lower) a race O (Tox1⁻) control. No significant differences in T-toxin production (indicated by halo sizes) among the three strains were evident.

Figure 2

Mutant R.C4.2696 has normal growth (A) and appressorium formation (B). (A) Plates containing complete medium with xylose (Upper) or minimal medium (Lower) were inoculated with mutant (Right) or WT (Left) and incubated in the light at 22°C for 7 days. (B) Mutant conidia (Lower) germinated and produced appressoria (arrows) like WT (Upper). Drops of conidial suspension (10⁵/ml in water) were placed on a glass slide in a humid chamber and incubated for 6 h at 32°C.

Figure 3

Targeted disruption of CPS1 in WT race T restored the original REMI mutant phenotype. (Upper) Corn plants (N cytoplasm) were inoculated with (Left to Right): REMI mutant R.C4.2696; WT race T (C4); WT race O (C5), and five race T disruptants obtained using linearized p214B7. (Lower) DNA gel blot showing that a single band (4.2 kb) is present in WT race T and race O (second and third lanes from Left), respectively, but is replaced by a 9.3-kb band (increased by the size, 5.1 kb, of pUCATPH) in all strains that showed the mutant phenotype in the plant assay shown (Upper). Genomic DNA was digested with BglII and probed successively with 5′ end and 3′ end flanking DNA fragments carried on p214B7 (strain order is the same as above).

Figure 4

Identification of CPS1. (A) Genomic DNA on both flanks of the REMI vector insertion site (solid arrowhead at KpnI site) in the mutant R.C4.2696 genome was recovered by two successive cycles of plasmid rescue. A partial restriction enzyme map of the 11.3-kb region around the tagged site is shown. The asterisks indicate two sites (HindIII and SacI) of vector integration, used for subsequent plasmid rescue. Locations of five rescued clones used for sequencing are shown by thin lines (triangle represents the position of pUCATPH in each clone). The ORF (ORF1 = CPS1) is indicated by the open arrow. Vertical bars in ORF1 indicate intron positions. (B) Domain organization of CPS1 and several of the top blast hits listed in Table 1. ORFs are indicated by thick black lines (drawn to scale with a maximum of 2,000 aa; the C terminus of BlmVI is not shown). AMP, AMP-binding; T, thiolation (phosphopantetheine attachment site); C, condensation. CPS1 and all hypothetical proteins have two AMP-binding domains (designated as CPS1A and CPS1B), both of which are similar to the AMP domain of the first modules of BlmVI and SafB.

Figure 5

Phylogenetic analysis of CPS1. (A) CPS belongs to the adenylate-forming enzyme superfamily, as shown in this simplified rendering, which highlights the relationships of the major enzyme families. Numbers are percentages of bootstrap support for each clade. See also Fig. 10. (B) CPS phylogeny showing an inferred duplication event resulting in CPS1 and CPS2 in ascomycetes. Shown is one most-parsimonious tree: 14 genes, 6,294 steps, 482 excluded characters, and 1,690 included characters, of which 1,244 are informative, 395 are uninformative, and 51 are constant. Rescaled consistency index = 0.653. Numbers above branches are amino acid changes; numbers below branches are bootstrap percentages estimating support for each branch.

Figure 6

Disruption of C. victoriae CPS1 causes reduced virulence. Inoculations: (Left to Right) WT (HvW), Tx7, Tx2, Tx9, and water. Oat seedlings inoculated with WT or Tx9 (an ectopic transformant) were completely killed; those inoculated with Tx7 and Tx2 (disruptants) showed a mixture of dead (white arrow) and healthy plants.

Figure 7

Disruption of G. zeae CPS1 causes reduced virulence. Left to Right: WT, Tx4, Tx10, TxH1, Tx11, and water. Wheat heads inoculated with WT or TxH1 (ectopic transformant) were completely bleached, whereas those inoculated with Tx4, Tx10, and Tx11 (disruptants) showed minor symptoms.