A Poplar Rust Effector Protein Associates with Protein Disulfide Isomerase and Enhances Plant Susceptibility

Abstract

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

Differential alteration of plant functions by homologous fungal candidate effectors

“…Melampsora larici-populina CEs have been previously studied in heterologous systems for functional characterization [19, 22, 25, 30, 31]. In Table 1, we present features of the 14 CEs studied here.…”

“…Mlp37347 is a homolog of the well studied AvrL567 group from M. lini [32, 33], and accumulates at the plasmodesmata in Arabidopsis . Mlp72983 accumulates in the chloroplast [19] and Mlp124357 is found in the tonoplast and was shown to interact with Arabidopsis and poplar Protein Disulfide Isomerase [30]. The other 11 CEs selected here have nucleocytosolic accumulation, the same as the marker protein GFP used.…”

“…Arabidopsis thaliana transgenic plants in Columbia-0 background expressing GFP alone (control) or fused to a candidate effector of the fungus Melampsora larici-populina (Mlp37347, Mlp72983, Mlp102036, Mlp106078, Mlp123218, Mlp123227, Mlp123531, Mlp124256, Mlp124266, Mlp124357, Mlp124466, Mlp124497, Mlp124499, Mlp124518) previously obtained in our laboratory [19, 30], were grown at 22°C at 12h/12h light/dark cycles.…”

“…Although the pathogen growth readout is informative regarding the impairment of the immune pathway, it is opaque with regards to which pathway has been tampered with or which metabolites are off-balance. Transcriptomic and metabolomic studies of stable transgenic plants expressing CEs have been useful in these cases, since they allow the detection of more subtle changes, unlikely to have a quantifiable impact on pathogen growth on their own [22, 29, 30].…”

“…Discussion Effector biologists have tackled both the identification and the functional characterization of candidate effectors (CEs) [14, 35], as this is a key step towards a better understanding of plant-microbe interactions. In rust fungi, different approaches are used in the functional characterization of these proteins, including analysis of subcellular localization in planta[19,25,28,30,31], infection assays in true host or in a model plant, and induction/repression of plant cell death[19,30,36,37]. The transcriptome or metabolome of the host in responses to the pathogen are frequently evaluated[38][39][40][41][42][43], but the assessment of the role of individual CEs in these processes is not easily measured and seldom analyzed[22,44].Here we investigated 14 CEs from Melampsora larici-populina by evaluating their individual impact on the transcriptome and metabolome of stable transgenic Arabidopsis plants.…”