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

Abstract

Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

“…For this, the promoter of the P. syringae effector gene AvrRpm1 (Upadhyaya et al, 2014) followed by a Gateway cassette, sfGFP11, HA epitope tag, and AvrRpm1 terminator was introduced into the broad-host-range vectors pBBR1MCS-2 and -5 (Kovach et al, 1995) ( Figure 4B). To deliver effectors from other pathogens that do not use T3SS, we engineered vectors with the AvrRpm1 promoter followed by a T3SS signal peptide (Upadhyaya et al, 2014). Since a very small amount of the effectors might be delivered from bacteria, we generated vectors containing 2xsfGFP11 to improve the fluorescence signal ( Figure 4B).…”

“…For the expression vector of the bacterial T3E with sfGFP11, broadhost-range vector, pBBR1MCS-2 and -5 (Kovach et al, 1995) were modified to introduce AvrRpm1 promoter (Upadhyaya et al, 2014) followed by the Gateway cassette with AvrRpm1 terminator. The series of sfGFP11 and linker were amplified using synthetic oligos and the corresponding fragments were introduced after the Gateway cassette in frame (Supplemental Figure 1B).…”

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.) (Retracted)

“…Pseudomonas spp. have been used as a delivery vehicle in Arabidopsis thaliana to characterize oomycete effector proteins by fusion to the type III signals of avirulence proteins AvrRps4 and AvrRpm1 (Rentel et al 2008;Sohn et al 2007); this system was also recently used in wheat to investigate effector candidates from the stem rust fungus Puccinia graminis tritici (Upadhyaya et al 2014). Additionally, Sharma and colleagues (2013) have employed the Burkholderia glumae T3S system to transfer Magnaporthe oryzae effectors to rice cells.…”

New insights in the battle between wheat and Puccinia striiformis

“…viciaefabae [90] , and PGTAUSPE-10-1 in the wheat stem rust fungus Pgt [91] . All of these proteins are thought to be translocated from haustoria to the host cell.…”

“…Immunocytological and biochemical studies indicated that RTP1 is a member of a new class of structural effectors, which are capable of forming filamentous structures in vitro and in vivo [93] . Due to the induction of cell death in the host line carrying the resistance gene Sr22, PGTAUSPE-10-1 was considered as a candidate AvrSr22 [91] . Apart from the six rust effectors, only a few secreted proteins have been cloned as candidate effectors on the basis of expressed sequence tags and cDNA library from Pst.…”