A functionally conserved Zn2 Cys6 binuclear cluster transcription factor class regulates necrotrophic effector gene expression and host-specific virulence of two major Pleosporales fungal pathogens of wheat

Abstract

The fungus Parastagonospora nodorum is the causal agent of Septoria nodorum blotch of wheat (Triticum aestivum). The interaction is mediated by multiple fungal necrotrophic effector-dominant host sensitivity gene interactions. The three best-characterized effector-sensitivity gene systems are SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. These effector genes are highly expressed during early infection, but expression decreases as the infection progresses to tissue necrosis and sporulation. However, the mechanism of regulation is unknown. We have identified and functionally characterized a gene, referred to as PnPf2, which encodes a putative zinc finger transcription factor. PnPf2 deletion resulted in the down-regulation of SnToxA and SnTox3 expression. Virulence on Tsn1 and Snn3 wheat cultivars was strongly reduced. The SnTox1-Snn1 interaction remained unaffected. Furthermore, we have also identified and deleted an orthologous PtrPf2 from the tan spot fungus Pyrenophora tritici-repentis which possesses a near-identical ToxA that was acquired from P. nodorum via horizontal gene transfer. PtrPf2 deletion also resulted in the down-regulation of PtrToxA expression and a near-complete loss of virulence on Tsn1 wheat. We have demonstrated, for the first time, evidence for a functionally conserved signalling component that plays a role in the regulation of a common/horizontally transferred effector found in two major fungal pathogens of wheat.

Keywords: Parastagonospora nodorum; Pyrenophora tritici-repentis; Septoria nodorum blotch; SnTox3; ToxA; effector regulation; tan spot.

Figure 1

A bootstrap consensus phylogenetic tree showing the amino acid relationships between Pf2 orthologues and other putative transcription factors from various plant‐pathogenic fungi. ScMal4 from Saccharomyces cerevisiae was used as an outgroup. Percentage bootstrap values from 1000 repetitions are shown above the branches. Italicized prefix abbreviation of species: Ab, Alternaria brassicicola; Bc, Botrytis cinerea; Bgh, Blumeria graminis f. sp. hordei; Bgt, Blumeria graminis f. sp. tritici; Cc, Cochliobolus carbonum; Cgl, Colletotrichum gloeosporioides; Cgr, Colletotrichum graminicola; Ch, Cochliobolus heterostrophus; Cm, Cochlioblus miyabeanus; Cs, Cochliobolus sativus; Cv, Cochliobolus victoriae; Ds, Dothiostroma septosporum; En, Erysiphe necator; Fg, Fusarium graminearum; Mf, Mycosphaerella fijiensis; Mo, Magnaporthe oryzae; Pn, Parastagonospora nodorum; Ptr, Pyrenophora tritici‐repentis; Ptt, Pyrenophora teres‐teres; Sb, Sclerotinia borealis; Sc, Saccharomyces cerevisiae; St, Setosphaeria turcica; Zt, Zymoseptoria tritici. Amino acid sequences are deposited as Data S1. Where applicable, strains are indicated in parentheses. * and ** denote differences in annotation between M4 and Pt‐1C_BFP orthologues. ^{^}Manually‐curated open reading frame (ORF) (Joint Genome Institute database).

Figure 2

Pf2 and effector genes are maximally expressed during early infection. PnPf2 (A) and SnToxA, SnTox1 and SnTox3 (B) in Parastagonospora nodorum gene expression profile in planta (n = 3). Parastagonospora nodorum‐infected wheat tissues are shown at the time of sampling. (C) Real‐time polymerase chain reaction (PCR) analysis of SnToxA, SnTox1, SnTox3, PnPf2 and Act1 expression in SN15 during growth in Fries 3 broth. In planta gene expression profile of PtrPf2 (D) and PtrToxA (E) (n = 3). Pyrenophora tritici‐repentis‐infected wheat tissues are shown at the time of sampling. Error bars are shown as the standard error of the mean in all graphs. (F) Real‐time PCR analysis of PtrToxA, PtrPf2 and PtrAct1 expression in P. tritici‐repentis during growth in Fries 3 broth. The experiment was performed using pooled biological triplicate samples.

Figure 3

Nucleotide alignment of the putative promoter region of ToxA (yellow) from Parastagonospora nodorum (SN15 and SN4) and Pyrenophora tritici‐repentis (M4 and Pt‐1C‐BFP) showing extensive nucleotide polymorphism at the putative 5′ promoter region. Nucleotide sequences are described in Fig. S3 (see Supporting Information).

Figure 4

PnPf2 is associated with host‐specific virulence on wheat. A whole‐plant infection assay on wheat cultivars possessing Tsn1, Snn1 or Snn3 genotypes. (A) Development of necrosis after 7 days post‐infection (dpi) and (B) pycnidia after 14 dpi. Genetic complementation restored virulence on BG261 and BG220 comparable with that on SN15 (Fig. S6).

Figure 5

PnPf2 is a positive regulator of SnToxA and SnTox3 expression. (A) Chlorotic necrotic symptoms of wheat cv. Calingiri (Snn1) and BG220 (Snn3) infiltrated with culture filtrates of SN15 and pf2‐69. Infiltration of BG261 (Tsn1) with culture filtrates of all strains did not result in chlorosis (data not shown). (B) Wheat cv. Halberd is susceptible to SN15 and pf2‐69. At 3 days post‐infection (dpi), trypan blue staining revealed that pf2‐69 attempts to enter the host through the stomata and epidermis via hyphopodia, similar to SN15 (red arrows). At 8 dpi, the necrotic lesion caused by pf2‐69 is comparable with that of SN15. (C) Halberd was used as a host to determine the expression of SnToxA, SnTox1 and SnTox3 in SN15 and pf2‐69 at 3 dpi. –, Tween non‐infection control. Error bars are shown as the standard error of the mean. Quantitative real‐time polymerase chain reaction was performed with biological triplicates.

Figure 6

SnToxA and SnTox3 pre‐infiltration restores virulence. Detached leaf assay of BG261 (Tsn1) (A) and BG220 (Snn3) (B) infected with SN15, Ect and pf2‐69 with and without effector pre‐infiltration. Pycnidiation is an indication of virulence in the effector pre‐infiltrated treatments (+).

Figure 7

SnTox1 deletion in a pnpf2 background abolishes virulence on Snn1 wheat. (A) SnTox1 and PnPf2 are required for virulence on Calingiri (tsn1, Snn1, snn3). (B) Quantitative real‐time polymerase chain reaction analysis of SnTox3 expression during in vitro growth. SnTox1 deletion does not restore SnTox3 expression in the pf2 background. Standard error bars are shown. Normalized SnTox3/Act1 values are provided above the error bars. Levels not connected by the same letter are significantly different (P < 0.05) based on analysis of variance. The experiment was performed with biological replicates (n = 6).

Figure 8

PtrPf2 regulates the production of PtrToxA and an undiscovered necrosis‐inducing factor(s). Culture filtrate activity (A) and virulence (B) of Pyrenophora tritici‐repentis M4 wild‐type, E‐1 and PtrPf2 deletion mutants on wheat cv. BG261 (Tsn1). Culture filtrate activity (C) and virulence (D) of P. tritici‐repentis M4 wild‐type, E‐1 and PtrPf2 deletion mutants on wheat cv. Machete (tsn1).