Prospects for functional analysis of effectors from cereal rust fungi

Abstract

With the advancement of several Puccinia genome sequencing projects, along with gene expression data and methods for predicting secreted proteins, it is now possible to predict many effector proteins from the cereal rusts. Biological assays that can be conducted in a relatively high throughput fashion are necessary to assign specific functions, such as avirulence. Biolistic delivery of potential effectors is limited by the need to examine individual cells and delivery by Agrobacterium generally also affects small numbers of cells in grasses. An approach that has had some success in dicots is the use of bacterial systems to deliver proteins by their type III secretion systems (TTSS). Several bacterial systems were thus tested for their suitability in delivering effectors to wheat. Pseudomonas syringae DC3000 caused hypersensitive reactions (HR) when infiltrated into all tested wheat lines but only some barley lines. A variant strain with multiple effectors deleted showed a reduced HR on wheat lines. Pseudomonas fluorescens with an engineered TTSS system showed no HR in wheat lines but was able to deliver bacterial effectors AvrRpm1 and AvrRpt2 and the fungal toxin ToxA. Delivery of the effectors by P. fluorescens could be detected by HR or by staining for presence of hydrogen peroxide or callose deposits. The bacterial systems thus showed good potential for their ability to deliver foreign proteins into wheat cells.

PSTha5a23, a candidate effector from the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici, is involved in plant defense suppression and rust pathogenicity

“…Recently, a bacterial type III secretion system (T3SS) assay for delivery of fungal effectors into wheat, using the expression/delivery vector pEDV6 and Pseudomonas fluorescens effector‐to‐host analyzer (EtHAn) strain, has been established (Yin and Hulbert, ; Upadhyaya et al ., ). Hence, we overexpressed PSTha5a23 in wheat cv.…”

“…Avocet Susceptible (AvS) using this assay to further investigate the role of PSTha5a23 in plant immunity modulation. eGFP and bacterial effector AvrRpt2 that can trigger HR in wheat (Innes et al ., ; Yin and Hulbert, ) were used as a negative and positive control respectively. EtHAn is non‐pathogenic on wheat (Yin and Hulbert, ) and inoculation of EtHAn in wheat could not trigger a detectable necrotic or chlorotic reaction phenotype on wheat plants (Fig.…”

Genome analyses of the wheat yellow (stripe) rust pathogen Puccinia striiformis f. sp. triticireveal polymorphic and haustorial expressed secreted proteins as candidate effectors

“…This crucial phase is still in its infancy [38,39] and will most likely be limited to testing a handful of candidate genes in the initial stages. Several groups have attempted to modify existing heterologous expression systems from other pathosystems to establish a reliable method for testing rust AVR effector candidates in wheat.…”

“…Several groups have attempted to modify existing heterologous expression systems from other pathosystems to establish a reliable method for testing rust AVR effector candidates in wheat. This includes delivery of effector candidates directly into wheat cells by expressing them in bacterial pathogens for delivery by the type III secretion system [39] or through virus-mediated approaches [40]. Alternatively, host-induced gene silencing could be utilized to transiently silence and test PST pathogenicity or virulence genes.…”

A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat

“…syringae 61 hrp/hrc cluster (T3SS machinery) stably integrated into the chromosome, thereby making this nonpathogenic bacterium T3SS competent for effector delivery. This was shown to function for delivery of various P. syringae effectors into A. thaliana (Thomas et al 2009), andYin andHulbert (2010) found that P. fluorescens EtHAn expressing AvrRpm1 or AvrRpt2 induced H 2 O 2 production in wheat, suggesting that it may be suitable for effector delivery in cereals. In this study, we report the development of an efficient system of effector delivery into wheat, based on T3SS delivery by P. fluorescens EtHAn, which has enabled the screening of candidate Puccinia graminis f. sp.…”