A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice

Abstract

Isolates of the rice blast fungus Magnaporthe grisea that carry the gene encoding Avirulence Conferring Enzyme1 (ACE1) are specifically recognized by rice (Oryza sativa) cultivars carrying the resistance gene Pi33. This recognition enables resistant plants to activate a defense response. ACE1 was isolated by map-based cloning and encodes a putative hybrid between a polyketide synthase and a nonribosomal peptide synthetase, enzymes involved in microbial secondary metabolism. ACE1 is expressed exclusively during fungal penetration of host leaves, the time point at which plant defense reactions are triggered. Ace1 appears to be localized in the cytoplasm of the appressorium. Mutation of the putative catalytic site of the beta-ketoacyl synthase domain of Ace1 abolishes recognition of the fungus by resistant rice. This suggests that Ace1 biosynthetic activity is required for avirulence. Our results are consistent with the hypothesis that the fungal signal recognized by resistant rice plants is the secondary metabolite whose synthesis depends on Ace1.

Figure 1.

Isolation of ACE1 by Complementation. Schematic representation of the ACE1 locus in Guy11, cosmids, and subclones tested for complementation to avirulence. The genomic locus is represented as a solid line, and the positions of RAPD marker OPE-Y13 and relevant restriction sites are indicated as follows: E, EcoRI; X, XbaI; B, BamHI; N, NotI; Bg, BglII. The ACE1 ORF is indicated as an arrow, and interruptions indicate positions of the three introns. Above the genomic locus map, the relative positions of the noncomplementing cosmid D22F11 and the complementing cosmid D31C12 are shown. Below the genomic locus map, the relative positions of D31C12 subclones used for complementation are shown. Fractions given to the right of each construct indicate the number of avirulent transformants identified over the number of transformants tested. Numbers without asterisks represent results obtained with virulent parent 2/0/3, numbers with asterisks represent results obtained with virulent field isolate PH14. Cosmid D31C12 and plasmid pEco20 complement both virulent strains to avirulence, demonstrating that they carry ACE1.

Figure 2.

Organization of Avirulence Gene ACE1 and Analysis of Its Partial Deletion Mutants. (A) Physical map of the complementing plasmid pEco20, insertion site of retroposon in 2/0/3 ace1 virulent allele, the gene replacement vector pΔACE1, and Ace1 protein encoded by ACE1. The ACE1 ORF is represented as a hatched box, the three introns (i1 to i3) as black bars, and restriction sites as E for EcoRI and A for AgeI. In the ace1 allele from virulent parent 2/0/3, a 2-kb sequence is inserted at 2.6 kb before the ACE1 stop codon indicated by a retroposon sign on the pEco20 physical map. In the gene replacement vector pΔACE1, the hph resistance gene (open box) replaces an internal 3.6-kb AgeI fragment. In the ACE1 translation product (Ace1), stippled boxes represent enzymatic domains (drawn to scale): KS, β-ketoacyl synthase; AT, acyltransferase; DH, dehydratase; MT, methyl-transferase; KR, β-ketoreductase; ACP, acyl carrier protein; C, condensation domain; A, AMP binding domain; PCP, peptidyl carrier protein. PKS designates the polyketide synthase part of Ace1 and NRPS the nonribosomal peptide synthetase module of Ace1. aa, amino acids. (B) Infection assays on resistant rice cultivar C101lac. Seedlings were spray inoculated with avirulent parent Guy11, virulent parent 2/0/3, 2/0/3 transformant HBE20 carrying pEco20, and Guy11 ace1 deletion mutant HB26. Typical symptoms on rice leaves were obtained 7 d after inoculation. Guy11 is unable to infect the Pi33-carrying rice cultivar C101lac. Isolate 2/0/3 is virulent on C101lac and induces typical susceptible lesions. The 2/0/3 transformant HBE20 is avirulent on C101lac as a result of the introduction of ACE1 carried by pEco20. HB26 is virulent on C101lac as a result of the partial deletion of ACE1 by gene replacement using the pΔACE1 construct.

Figure 3.

Phylogeny of Fungal PKS Based on Protein Sequence of KS and AT Domains. The consensus phylogenetic tree was obtained using the neighbor-joining method with distances. Bootstrap values are indicated above or below nodes of the tree. Colored boxes represent PKS and NRPS enzymatic domains (see Figure 2 for definitions). Sequences from the following organisms were included in this analysis (see Methods for accession numbers): M. grisea (Ace1, Syn2, Syn6,7,8, mg03810, mg03818, and mg09589); A. fumigatus (af04917); A. nidulans (AN8412, AnpksST, and AnWA); F. graminearum (FG10464); N. crassa (ncu08399, ncu04865, ncu09638, and ncu02918); A. terreus (LNKS, LDKS, AtMSAS, and Atat1); P. citrinum (MlcA); P. griseofulvum (PgPKS2), C. heterostrophus (ChPKS1); G. moniliformis (FUM1), and C. lagenarium (ClPKS1). We added fungal PKS and PKS/NRPS sequences described by Kroken et al. (2003) from C. heterostrophus (ChPKS2,16,17), B. fuckeliana (BfPKS3,4,5,6,8), G. moniliformis (GmPKS1,9,10), and G. zeae (GzPKS10) and bacterial PKS sequences (epoD, MxaC, and MxaD) as references. FAS from the silk moth Bombyx mori was included as an outgroup to root the tree and PKS/NRPS cluster as a monophyletic group. *, Although GzPKS10 is only described as a partial PKS sequence, its relatedness to GmPKS10 (90% identity) suggests that it has a full NRPS module as GmPKS10. **, FG10464 is almost identical (>91% identity) to GzPKS9 from Kroken et al. (2003). ncu08399 is identical to NcPKS4 from Kroken et al. (2003). LNKS is identical to lovB from Kroken et al. (2003).

Figure 4.

Expression Pattern of ACE1 in Avirulent Isolate Guy11. (A) PCR and RT-PCR amplification of the constitutively expressed ACT1 gene as control of RNA integrity. The region spanning the first three introns of ACT1 was amplified. Lane 1, ACT1 PCR product obtained from genomic DNA (size, 900 bp). Lane 2, ACT1 RT-PCR product obtained from RNA isolated from barley leaves infected with Guy11 17 h after infection (size, 350 bp). Lane 3, ACT1 RT-PCR product obtained from RNA isolated from Guy11 mycelial culture. The amplification products were separated by agarose gel electrophoresis and stained with ethidium bromide. (B) PCR and RT-PCR amplification of ACE1 from avirulent parent Guy11. The region spanning the first intron of ACE1 was amplified. Lane 1, ACE1 PCR product obtained from genomic DNA (size, 453 bp). Lane 2, ACE1 RT-PCR product obtained from RNA isolated from barley leaves infected with Guy11 17 h after inoculation (size, 365 bp). Lane 3, No ACE1 RT-PCR product was obtained with RNA isolated from Guy11 mycelial culture, suggesting that ACE1 is not expressed in mycelium.

Figure 5.

Localization of Ace1-GFP Fusion Protein in M. grisea Appressoria. (A) GFP fluorescence of appressoria and infectious hypha after penetration of barley leaves. Conidia of transformant IF22 expressing GFP under the control of the ACE1 promoter were inoculated on detached barley leaves and incubated at 26°C. At 36 h after infection, epidermal strips were removed from the infected leaf and observed under a microscope. Under blue light, GFP fluorescence is detected both in appressorium and in infectious hyphae. Bar = 10 μm. (B) Same view as (A) under bright field. An appressorium and its infectious hypha located in the underlying epidermal cell are visible. (C) Ace1-GFP fluorescence of appressoria on the surface of barley leaves. Conidia of transformant HB41 expressing the Ace1-GFP fusion protein under the control of ACE1 promoter were inoculated on detached barley leaves incubated for 24 h at 26°C and observed with confocal laser scanning microscopy. Under blue light, fluorescence of the Ace1-GFP fusion protein is detected exclusively in the cytoplasm of the appressoria. Bar = 10 μm. (D) Same view as (C) under bright field. Two germinated spores and appressoria are visible on the leaf surface. (E) Ace1-GFP fluorescence of appressoria after penetration of barley leaves. Conidia of transformant HB44 expressing the Ace1-GFP fusion protein were inoculated on detached barley leaves and incubated at 26°C. Epidermal strips were removed from the infected leaf 40 h after inoculation and observed under a microscope. Under blue light, GFP fluorescence is detected in the appressorium but not in infectious hypha. Bar = 10 μm. (F) Same view as (E) under bright field. An appressorium and its infectious hypha located in the underlying epidermal cell are visible.

Figure 6.

Lack of Avirulence Conferring Activity of the ace1^C183A Allele. (A) Conidia of the transformants HB41 and HB10 were inoculated on detached barley leaves and incubated at 26°C. Epidermal strips were removed from the infected leaf 20 h after infection and observed under a microscope. Appressoria were observed under bright-field illumination (left) or UV light (right). The GFP fluorescence of Ace1-GFP and Ace1^C183A-GFP provides a control of the expression of the transgenes in appressoria and the correct localization of their respective translation product. Expression and localization of Ace1-GFP (HB41) and Ace1^C183-GFP (HB10) were similar, indicating that the Ace1^C183-GFP protein is expressed and localized as wild-type Ace1 protein. Bars = 8 μm. (B) Avirulent isolate Guy11, virulent parent 2/0/3, and transformants of 2/0/3 expressing the ACE1:GFP fusion (HB41) or the mutant ace1^C183A:GFP allele (HB10) were inoculated on resistant rice cultivar C101lac. Guy11 and HB41 carrying ACE1:GFP are avirulent. Transformant HB10 and 2/0/3 carrying the mutant ace1^C183A:GFP allele are virulent.