Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses.

Mingsheng Qi,Ralf T Voegele,Janina M Seitz,Luana M Darben,James P Grayczyk,Steven A Whitham,Yu Mei,Tobias I Link,Martin E G Rieker

doi:10.3389/fpls.2019.01182

Abstract

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.

Highlights

Fungal pathogens use effectors to influence their host plants
GFP-Uaca_1, was mostly located in the nucleus (Figure 4A). These findings indicate that U. appendiculatus, similar to other rust fungi, may deliver effector proteins to a variety of distinct host cell compartments (Petre et al, 2015)
Little is known about the functions of effectors produced by rust fungi

Summary

Introduction

Fungal pathogens use effectors to influence their host plants. These effectors may function in weakening or killing the infected tissue as is the case for necrotrophic pathogens. We described a comprehensive pipeline of assays in heterologous systems that aimed to identify true effectors from collections of candidate effectors (Qi et al, 2018). This pipeline is centered on identifying candidate effectors that suppress plant defense responses. The bacterial type III secretion system (T3SS), which has the natural purpose of transferring effector proteins into host cells, is a well-established approach for testing the ability of fungal effectors to suppress defense responses (Sohn et al, 2007). If leaf tissue, which was first infiltrated with P. fluorescens EtHAn carrying an effector candidate and later with Pst DC3000, shows HR, this can be considered evidence that the effector can suppress basal defense

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Conclusion