A ToxA-like protein from Cochliobolus heterostrophus induces light-dependent leaf necrosis and acts as a virulence factor with host selectivity on maizeFungal Genetics and Biology

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Authors
Shunwen Lu, B. Gillian Turgeon, Michael C. Edwards
Year
2015
DOI
10.1016/j.fgb.2015.05.013
Subject
Genetics / Microbiology

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er a aUS Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Fargo, ND 58102-2765, USA b Plant Pathology & Plant-Microbe Biology Section, Scho a r t i c l e i n f o

Article history:

Received 29 April 2015

Revised 28 May 2015

Accepted 29 May 2015

Available online 4 June 2015 1. Introduction that is essential for high virulence on maize lines with Texas male-sterile cytoplasm (Yang et al., 1996), HC-toxin, a cyclic ace 1, that condirecessive at the hat detox chlorinated that accou et al., 1985). Collectively, these well-characterized HSTs are small molecule secondary metabolites biosynthesized independently of ribosomes, by large multidomain megaenzymes such as polyketide synthases or nonribosomal peptide synthetases.

In addition to secondary metabolite HSTs, some HSTs are produced ribosomally. The first reported fungal proteinaceous

HST (ToxA) was isolated from the tan spot fungus Pyrenophora tritici-repentis (Ptr) about 25 years ago (Ballance et al., 1989; q Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. ⇑ Corresponding author. Tel.: +1 (701) 239 1344; fax: +1 (701) 239 1369.

E-mail address: Shunwen.Lu@ars.usda.gov (S. Lu).

Fungal Genetics and Biology 81 (2015) 12–24

Contents lists availab

Fungal Genetic journal homepage: www.eTurgeon and Lu, 2000; Condon et al., 2013). Examples include

T-toxin, a polyketide toxin produced by C. heterostrophus race T

Victoria blight on oats carrying the dominant Pc-2 allele introduced for resistance to crown rust caused by Puccinia coronata (MackoThe maize pathogen Cochliobolus heterostrophus and closely related species in the genus are best known for producing host-selective toxins (HSTs) associated with ability of the fungus to cause severe diseases on host cereal crops (Yoder, 1980; tetrapeptide produced by Cochliobolus carbonum r tions pathogenicity on maize lines homozygous

Hm1/Hm2 loci encoding carbonyl reductases t toxin (Panaccione et al., 1992), and victorin, a pentapeptide produced by Cochliobolus victoriaehttp://dx.doi.org/10.1016/j.fgb.2015.05.013 1087-1845/Published by Elsevier Inc.ify the cyclic nts fornew insights into effector evolution in host–pathogen interactions.

Published by Elsevier Inc.Keywords:

Host-specific toxin

Necrotrophic effectors

Southern corn leaf blight

Fungal pathogenicity

Effector-trigged susceptibilityol of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA a b s t r a c t

ToxA, the first discovered fungal proteinaceous host-selective toxin (HST), was originally identified in 1989 from the tan spot fungus Pyrenophora tritici-repentis (Ptr). About 25 years later, a homolog was identified in the leaf/glume blotch fungus Stagonospora nodorum (Parastagonospora nodorum), also a pathogen of wheat. Here we report the identification and function of a ToxA-like protein from the maize pathogen

Cochliobolus heterostrophus (Ch) that possesses necrosis-inducing activity specifically against maize.

ChToxA is encoded by a 535-bp open reading frame featuring a ToxA-specific intron with unusual splicing sites (50-ATAAGT. . .TAC-30) at conserved positions relative to PtrToxA. The protein shows 64% similarity to

PtrToxA and is predicted to adopt a similar three-dimensional structure, although lacking the arginyl-glycyl-aspartic acid (RGD) motif reported to be required for internalization into sensitive wheat mesophyll cells. Reverse-transcriptase PCR revealed that the ChTOXA gene expression is up-regulated in planta, relative to axenic culture. Plant assays indicated that the recombinant ChToxA protein induces light-dependent leaf necrosis in a host-selective manner on maize inbred lines. Gene deletion experiments confirmed that ChtoxA mutants are reduced in virulence on specific ChToxA-sensitive maize lines, relative to virulence caused by wild-type strains. Database searches identified potential ChToxA homologues in other plant-pathogenic ascomycetes. Sequence and phylogenetic analyses revealed that the corresponding ToxA-like proteins include one member recently shown to be associated with formation of penetration hypha. These results provide the first evidence that C. heterostrophus is capable of producing proteinaceous HSTs as virulence factors in addition to well-known secondary metabolite-type toxins produced biosynthetically by polyketide synthase megaenzymes. Further studies on ChToxA may provideShunwen Lu a,⇑, B. Gillian Turgeon b, Michael C. Edwards aRegular Article

A ToxA-like protein from Cochliobolus het light-dependent leaf necrosis and acts as with host selectivity on maizeqostrophus induces virulence factor le at ScienceDirect s and Biology lsev ier .com/ locate/yfgbi 2006) in the leaf/glume blotch fungus Parastagonospora nodorum (previously known as Stagonospora nodorum [Sn], and used s andhereafter for convenience) (Friesen et al., 2006). Subsequently an ortholog was also discovered in Stagonospora avenaria tritici 1, a sister species of S. nodorum (McDonald et al., 2013). It has been proposed that ToxA was acquired by P. tritici-repentis from

S. nodorum through a lateral/horizontal gene transfer (Friesen et al., 2006). To date, no ToxA-like proteins have been characterized from other plant-pathogenic ascomycetes.

We recently reported the cloning of a ToxA-like gene (ChTOXA [ToxA in Ptr and Sn]) from C. heterostrophus and showed that it encodes a protein with both sequence and structure similarities to PtrToxA (Lu and Edwards, 2014). In this manuscript, we describe

ChTOXA and ChTOXA-like genes from C. heterostrophus and closely-related species and functional characterization of ChToxA by recombinant protein and gene deletion approaches. We demonstrate that the recombinant ChToxA protein induces light-dependent leaf necrosis in a host-selective manner and that the native ChTOXA gene product acts as a virulence factor on specific ChToxA-sensitive maize lines. Our study provides the first evidence that C. heterostrophus produces both proteinaceous and secondary metabolite-derived HSTs as virulence factors. We also present evidence that potential ToxA homologues exist in other plant pathogenic fungi including both Dothideomycete and