Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Abstract

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogenencoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…More importantly, the RxLR motif has been demonstrated to be required for cell entry without the presence of the pathogen by both oomycete and fungal effectors (11,118,(137)(138)(139), emphasizing its major role in this mechanism. Indeed, we observe that loss of an intact RFLR motif in Avh5 impairs internalization of the protein in human epithelial and soybean root cell entry assays ( Figure 36A, B, and 37).…”

Identification of Plasmodiophora brassicae effectors — A challenging goal

“…In C. fulvum , cys-rich effectors can inhibit and protect against plant hydrolytic enzymes, such as proteases, glucanases, and chitinases [32]. Cys-rich small-secreted proteins have also been identified as major effectors in the obligate biotrophic pathogens Melampsora larici-populina [35], and the Asian soybean rust fungus Phakopsora pachyrhizi [42], where one of the cys-rich small proteins identified as an effector has been shown to suppress plant immunity [43]. …”

“…The motif RxLR, arginine-any amino acid-leucine-arginine, has been identified in the N-terminal of some oomycete and fungal effectors [43,44]. Although the function of the RxLR motif in effector proteins remains unclear, it has been shown to be necessary for translocation into the host cell [45] and to elicit immune responses in plant cells [46].…”

Microbe-Independent Entry of Oomycete RxLR Effectors and Fungal RxLR-Like Effectors Into Plant and Animal Cells Is Specific and Reproducible

“…Two classes of experiments have provided information that these effectors do not require the microbe for entry. The first are plant transient expression experiments, by bombardment or agroinfiltration, in which the effector in question is secreted from the plant and then must re-enter to trigger a measured response (usually cell death in the presence of a cognate R gene) (Dou et al, 2008;Rafiqi et al, 2010;Gu et al, 2011;Anderson et al, 2012;Sun et al, 2013). The second is to expose root tips or leaf tissue to purified effectors and then, to measure entry microscopically using antibodies, or by the presence of a chemical or protein fluorescent label attached to the effector (Dou et al, 2008;Plett et al, 2011;Wawra et al, 2012).…”