Gene discovery and gene function assignment in filamentous fungi

Abstract

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Figure 1

Transposon-arrayed gene knockouts. The IVT reaction includes a transposon, the corresponding transposase, and recipient DNA. The transposon carries the hygromycin phosphotransferase gene that confers resistance to hygromycin in Escherichia coli, M. grisea, M. graminicola, and other filamentous fungi (35). The recipient DNA consists of cosmid clones. The cosmid vector contains homing endonuclease sites flanking the cloning site and the β-lactamase gene. The IVT products are transformed into E. coli, and individual transposon insertion sites are determined by sequencing. Cosmids containing an appropriate transposon insertion are digested with the homing endonuclease to release full-length inserts for transformation. Ectopic and TI events are distinguished by PCR analysis, and mutants are subjected to phenotype analysis. EI, ectopic integration.

Figure 2

TI is a function of homologous DNA length. TI frequency vs. homologous DNA length is plotted for three different data sets. ▴, Five published gene knockouts in M. grisea strain Guy 11. MA, MAC1 (36); P, PMK1 (23); A, ABC1 (37); M, MPG1 (21); N = NUT1 (38). Published TI frequencies are 16%, 11%, 21%, 3%, and 22%, respectively. □, MAC1 TAGKO cosmid subjected to KpnI, EcoRI and SpeI, restriction digestion yielding, three MAC1 constructs with the same insertion but different homologous DNA lengths of 5.5 kb, 13.5 kb, and 20.2 kb, respectively. ○, Ten different TAGKO constructs created by IVT into a cosmid library pool.

Figure 3

In vitro transposition site distribution. Comparison of transposition insertion site distribution in the MAC1 cosmid insert (≈36 kb) for transposons Thyra, Frigg, Sif, and Vor. The percentage of transposition events (y axis) within 1-kb segments (x axis) of the cosmid is shown. The number of insertion events (n) analyzed for each transposon is as follows: SIF, n = 89; Vor, n = 237; Frigg, n = 209; Thyra, n = 336.

Figure 4

Analysis of M. grisea TAGKO mutants in MAC1. (A) Transposon insertion sites in MAC1. P designates the probe used for RNA analysis. (B) Fungal growth assays. Mutant and WT strains were grown on oatmeal agar and photographed after 7 days. (C) Appressorium formation on various surfaces or in the presence of cAMP. The experiment was repeated three times with 250 germinated spores counted for each strain. (D) Rice pathogenicity assays. Sample leaves were photographed 7 days after inoculation. (E) RNA blot analysis of MAC1 mRNA levels. Thirty micrograms of total RNA was used for each strain.

Figure 5

Analysis of M. graminicola and M. grisea HPD4 TAGKO mutants. (A) The l-phenylalanine (l-phe) and l-tyrosine (l-tyr) degradation pathway. X indicates the pathway block by inactivation of HPD4. (B) M. graminicola and M. grisea HPD4 alleles showing the location of the TAGKO insertions. (C) Graphs showing growth of M. graminicola WT vs. IM+411 at 7 days in the presence of 95 different nitrogen sources (l-Phe and l-Tyr are indicated) and time courses of M. graminicola WT, IM-20, and IM+411 in MM-N + Phe and MM-N + Tyr, respectively. (D) Graphs showing growth of M. grisea WT vs. IM+840 at 7 days in the presence of 95 different nitrogen sources (l-Phe and l-Tyr are indicated) and time courses of M. grisea WT, IM-190, and IM+840 in MM-N + Phe and MM-N + Tyr, respectively. (E) Plate images showing growth recovery on complete medium of M. graminicola WT, IM-20, and IM+411 (after 8 days) and M. grisea WT, IM-190, and IM+840 (after 5 days) after incubation in MM, MM-N + Phe or MM-N + Tyr, as indicated. The schematic plate diagram depicts the location of each strain.