New capabilities for Mycosphaerella graminicola research

Abstract

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.

Figure 1

Wild‐type and transformed Mycosphaerella graminicola following 5 days of selection with G418 and carboxin. (A) Wild‐type (wt) and M. graminicola transformed with the neomycin phosphotransferase II (nptII) gene (G418^R), conferring resistance to G418, growing on increasing concentrations of G418. (B) Wild‐type (wt) and M. graminicola transformed with the MgSDHB(H267Y) gene (carboxin^R), conferring resistance to carboxin, growing on increasing concentrations of carboxin.

Figure 2

Disruption of the MgKU70 locus. (A) Diagram showing the construct used to disrupt the MgKU70 gene. (B) Diagram showing the native MgKU70 locus. (C) Diagram showing the disrupted MgKU70 locus after transformation in the correctly targeted strains. (D) Agarose gel showing the results of the verification polymerase chain reaction (PCR) carried out on 13 of the putative MgKU70 disruptant transformants (sample 14 is wild‐type untransformed DNA control, and sample 15 is a no‐template control for PCR). A product from the primer pair Ku70verF1/Ku70verR1 indicates the presence of the disruption cassette within a strain. A product from the primer pair Ku70verF2/Ku70verR2 indicates the presence of the wild‐type MgKU70 locus. As expected, the untransformed control only contains a product from Ku70verF2/Ku70verR2. Ectopic transformants result in products from both sets of primer pairs: samples 2–7 and 11–13. In MgKU70 gene disruptants, a product is obtained only with primers Ku70verF1/Ku70verR1: samples 1, 8, 9 and 10. (E) Southern blot analysis of three of the MgKU70 gene disruptant strains K4901 (T1), K4902 (T2) and K4903 (T3) and the wild‐type (wt) control K4418. The genomic DNA was digested with HindIII, and probed with a fragment of the selectable marker gene neomycin phosphotransferase II (nptII) (nptII region amplified as probe as indicated by a full black line in C). K4902 and K4903 contain single‐copy insertion events as a single band of 2.496 kb has hybridized with the probe. K4901 is an uncut control.

Figure 3

In vitro and in planta growth comparisons of the wild‐type and MgΔKU70 strains. (A) Growth of the wild‐type Mycosphaerella graminicola strain K4418 (wt) and two ΔMgKU70 strains K4902 and K4903 in Vogel's semi‐solid minimal medium. The values are averages for three replicates for each strain; bars represent the associated standard error of the mean. OD, optical density. (B) The percentage of disease coverage on wheat leaves infected with the wild‐type M. graminicola strain K4418 (wt) and the two ΔMgKU70 M. graminicola strains K4902 and K4903, 17 days post‐infection. The values are averages for 10 replica pots infected with each strain; bars represent the associated standard errors of the mean.

Figure 4

Targeted gene replacement at the MgTUB1 locus. (A) Diagram showing the construct used to replace the wild‐type MgTUB1 with a resistant allele. (B) Diagram showing the native MgTUB1 locus. (C) Diagram showing the replaced MgTUB1 locus after transformation in the correctly targeted strains. (D) Agarose gel showing the results of the polymerase chain reaction (PCR) verification of the MgTUB1 gene replacement transformants. Primers BtubHRverF2/hygromycin1 produce a 1577‐bp product for all successfully transformed strains. The primers BtubHRverF1/BtubHRverR1 amplify a product of 1925 bp when the construct successfully integrates at the MgTUB1 locus. The same primer pair amplifies a 501‐bp product when the wild‐type locus is retained, indicating ectopic transformants. Samples 1–8 were from wild‐type transformed M. graminicola and the transformants 1, 3 and 7 show correctly targeted gene replacement. Samples 9–16 were transformed into the ΔMgKU70 strain and all show correctly targeted gene replacement.

Figure 5

Targeted gene replacement at the MgERG11 locus. (A) Diagram showing the construct used to replace the wild‐type MgERG11 with a resistant allele. (B) Diagram showing the native MgERG11 locus. (C) Diagram showing the replaced MgERG11 locus after transformation in the correctly targeted strains. The primers used to verify the transformants in which the MgERG11 gene has been correctly replaced are shown. Primers erg11 fwd‐int/erg11 rev‐int amplify a product of 492 bp when the wild‐type MgERG11 locus is present and a 1876‐bp product in all transformants. Primers erg11 fwd‐ext/erg11 rev‐ext produce a product of 1750 bp only when correctly targeted gene replacement has occurred, but no product when an ectopic insertion is present.

Figure 6

Verification of the MgTUB1 gene replacement transformants containing the A198 allele. (A) Diagram showing the position of the primers used to amplify a fragment of the MgTUB1 gene, prior to restriction digestion of the polymerase chain reaction (PCR) product with the enzyme HgaI. Primers BtubHRverF4/BtubHRverR3 amplify a 501‐bp product in all transformants. The A198 mutation encodes an HgaI site not present in the wild‐type E198 allele and, when the PCR product is digested with HgaI, results in two diagnostic bands at 340 and 161 bp. (B) Agarose gel showing the HgaI digest of the PCR products for seven of the MgTUB1 gene replacement transformants. Sample 5 contains the E198 allele, as only the undigested 501‐bp product is present, where as samples 1–4, 6, 7 all have the desired A198 allele, the 501‐bp product is absent and the diagnostic 340‐bp and 161‐bp bands are present. As expected, the wild‐type control displays only the 501‐bp band, whereas an ectopic transformant shows all three bands.

Figure 7

Southern blot of the MgTUB1 gene replacement transformants transformed into the wild‐type (wt) (K4418) or ΔMgKU70 (K4903) background. Genomic DNA was digested with EcoRI and probed with a fragment of MgTUB1, which spans an internal EcoRI site. K4913 (T1), K4914 (T2) = A198 in ΔMgKU70, K4918 (T3), K4919 (T4) = E198 in ΔMgKU70, K4920 (T5), K4921 (T6) = E198 in wild‐type, K4922 (T7), K4924 (T8) = A198 in wild‐type. The ∼10‐kb band is upstream from the internal EcoRI site in the MgTUB1 probe and is common to transformed and untransformed strains. Untransformed wt and ΔMgKU70 strains display a 1.395‐kb band. T1–T8 all contain a single‐copy insertion of the MgTUB1 gene replacement cassette, indicated by a shift in size from 1.395 kb to 2.797 kb, corresponding to the presence of the hygR cassette. The ∼1.5‐kb band in all strains may result from hybridization to an MgTUB1 pseudogene (see main text for further details).

Figure 8

Homologous recombination at the MgTUB1 locus. (A) Diagram showing homologous recombination occurring in the 0.910 kb between the hygromycin resistance cassette and the A198 mutation, thus resulting in the incorporation of the E198 allele in the gene replacement transformant. (B) Diagram showing homologous recombination occurring in the 2.653 kb between the A198 mutation and the 5′ end of the DNA fragment, thus resulting in the incorporation of the A198 allele in the gene replacement transformant.

Figure 9

The percentage growth inhibition to benomyl of MgTUB1 gene replacement strains, generated in both the wild‐type and ΔMgKU70 parental background. K4903 = ΔMgKU70 strain (wt MgTUB1); K4418 (wt) = wild‐type; K4913 and K4914 = A198 in ΔMgKU70; K4918 and K4919 = E198 in ΔMgKU70; K4920 and K4921 = E198 in wild‐type background; K4922 and K4924 = A198 in wild‐type background. The average IC₅₀ value for the strains with the wild‐type E198 allele is 0.155 p.p.m. benomyl. All four strains with the A198 allele are not inhibited by 1000 p.p.m. benomyl, a resistance factor estimated to be >5000.