Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots

Abstract

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

Fig. 1.

The Mycosphaerella fijiensis Avr4 (MfAvr4) triggers a Cf-4– and Hcr9-Avr4–mediated hypersensitive response (HR). (A) Specific induction of HR by MfAvr4 in a MM-Cf-4 and a MM-Hcr9-4D tomato line transgenic for 35S::Hcr9-4D. Tomato plants were inoculated with Agrobacterium tumefaciens transformants expressing PVX::MfAvr4. HR was induced only in MM-Cf-4 and the MM-Hcr9-4D transgenic tomato line but not in the MM-Cf-0 line. As controls, the empty binary vector pSfinx (PVX) and the C. fulvum Avr4 (PVX::Avr4) were used. As expected, inoculation with PVX::Avr4 resulted in an HR only in MM-Cf-4 and the MM-Hcr9-4D transgenic tomato. Only mosaic symptoms caused by PVX were observed after inoculation of the three lines with the empty pSfinx vector. Photographs were taken 20 days post-inoculation. (B) Transient coexpression of Cf-4 and the Avr4 homologs from C. fulvum and M. fijiensis, in Nicotiana benthamiana by the Agrobacterium tumefaciens transient transformation assay (ATTA). For ATTAs, A. tumefaciens cultures were mixed in a 1:1 ratio and infiltrated in leaves of N. benthamiana plants. Coexpression of MfAvr4 and tomato Cf-4 or the Hcr9-Avr4 homologs from Solanum chmielewskii (Hcr9-Avr4-chm1), S. parviflorum (Hcr9-Avr4-par1), and S. peruvianum (Hcr9-Avr4-per1) results in HR in N. benthamiana leaves. As positive controls, C. fulvum Avr4 (Avr4) was coexpressed with Cf-4/Hcr9-Avr4s.

Fig. 2.

Induced hypersensitive response (HR) and necrosis in tomato after transient expression of the Mycosphaerella fijiensis Ecp2 homolog (MfEcp2), using the PVX-based expression system. Cotyledons of 2-week-old MM-Cf-Ecp2, MM-Cf-0, and MM-Cf-4 tomato plants were inoculated with Agrobacterium tumefaciens transformants expressing PVX::MfEcp2. As controls the empty binary vector pSfinx (PVX) and the C. fulvum Ecp2 (PVX::Ecp2) were used. Transient expression of MfEcp2 in MM-Cf-0 and MM-Cf-4 tomato lines resulted in induction of necrosis, while an additional HR is induced in the MM-Cf-Ecp2 line, as reflected by the higher level of necrosis. As expected, the PVX-based expression of C. fulvum Ecp2 (PVX::Ecp2) resulted in an HR only on the MM-Cf-Ecp2 line. Only mosaic symptoms caused by PVX were observed upon inoculations of the lines with the empty pSfinx vector. Photographs were taken 21 days post-inoculation.

Fig. 3.

Proposed functions for Ecp2 and Avr4. (A) In the absence of Cf resistance proteins, Ecp2 promotes virulence by interacting with an in planta target, causing host cell necrosis (indicated by the brown color of the host cell) that facilitates the necrotrophic mode of nutrition of hemibiotrophs, such as Mycosphaerella fijiensis. In case of biotrophs, such as Cladosporium fulvum coevolution between host and pathogen has fine-tuned Ecp2 to only weakly perturb the host cells without inducing cell necrosis. Avr4 is a defensive virulence factor, which interacts with fungal cell-wall chitin (indicated as an orange glow around the hyphae) to protect it against hydrolysis by host chitinases (4). (B) In the presence of cognate Cf resistance proteins, a hypersensitive response (HR) is induced that arrests fungal growth. In this model, we speculate that Cf-Ecp2 guards the virulence target of Ecp2 and triggers a Cf-Ecp2-mediated HR that is epistatic over virulence target-mediated necrosis. In contrast, Cf-4 presumably interacts directly with Avr4, and triggers an HR.