In planta expression screens of candidate effector proteins from the wheat yellow rust fungus reveal processing bodies as a pathogen-targeted plant cell compartment

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. Our work identifies P-bodies as a novel plant cell compartment targeted by pathogen effectors.

demonstrating that these regions are necessary for specific nuclear accumulation ( Figure 2B).  To gain further insight into the putative functions of the candidate effectors, we next associated with an average of 98 proteins, ranging from 20 to 236 ( Figure 3A). Conversely, a 1 5 0 plant protein associated with an average of 3.5 candidate effectors, ranging from 1 to 16 1 5 1 ( Figure 3B). Given the high complexity of the dataset, we used a scoring method we 1 5 2 previously developed to discriminate reliable and specific interactors (high score) from all specifically coimmunoprecipitated with a single candidate effector ( Figure 3C, Figure 4).

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For instance, the protein with the highest score (108) was an enhancer of mRNA decapping 1 5 7 protein 4 (NbEDC4) that specifically and robustly immunoprecipitated with PST02549 (Table   1 5 8 S2). Our coIP/MS assays showed that PST02549 specifically associated with NbEDC4. To evaluate the biological significance of this association, we first identified and cloned the acids) and exhibit a pairwise amino acid sequence identity of between 42 and 46% ( Figure   1 6 9 5A). The amino acid sequence identity between these proteins reaches 75% in the N-terminal 1 7 0 WD40 domain ( Figure 5B). Next, we expressed a TaEDC4 foci we observed were P-bodies. To test this hypothesis, we co-expressed TaEDC4-mCherry   1  7  4 with YFP-VCSc, a marker of P-bodies (Xu et al., 2006). Confocal microscopy revealed 1 7 5 perfectly overlapping signals in cytosolic foci, confirming that TaEDC4 accumulates in P-1 7 6 bodies in N. benthamiana leaf cells ( Figure 5C).

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To determine whether PST02549 and TaEDC4 associate in planta, we co-expressed performed anti-GFP coimmunoprecipitation followed by immunoblotting or sodium dodecyl the two proteins ( Figure 6). As negative controls, we used three GFP and three mCherry of experiments, we conclude that PST02539 and TaEDC4 specifically and robustly associate 1 9 0 in P-bodies in N. benthamiana leaf cells. During confocal microscopy assays of N. benthamiana leaf cells co-expressing 1 9 4 PST02549-GFP and TaEDC4-mCherry, we noted that the P-bodies appeared larger than 1 9 5 usual ( Figure 7A). To quantify this phenomenon, we measured the diameter of P-bodies from 1 9 6 confocal microscopy images. When PST02549-GFP was co-expressed with TaEDC4-1 9 7 mCherry or an untagged version of TaEDC4, the average diameters of the P-bodies were 4.5 1 9 8 ± 3.5 µm and 4.9 ± 2.2 µm, respectively ( Figure 7B, Table S4). By contrast, when PST02549-1 9 9 GFP and TaEDC4-mCherry were expressed independently and/or with other control proteins, with PST02549 (Table S5). None of the negative controls we tested co-localised with  and C, Table S4). We conclude that co-expression of PST02549 and TaEDC4 specifically 2 0 5 increases the size of P-bodies. In this study, we found that PST02549 accumulates in plant cell P-bodies and 2 0 9 associates with a P-body-derived protein. This observation suggests that an effector that 2 1 0 targets plant P-bodies has evolved in P. striiformis f sp tritici. To our knowledge, a connection 2 1 1 between filamentous plant pathogens and P-bodies has not previously been established.

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How would a pathogen benefit from manipulating host P-bodies? Some plant significance of this phenomenon.

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We observed an increase in P-body size upon co-expression of PST02549 and TaEDC4. The depletion or overexpression of P-body components is known to modify P-body 2 2 5 integrity, which can lead to an increase in size (Eulalio et al., 2007). It is therefore possible 2 2 6 that the increase in P-body size observed in our study is due to over-accumulation of P-body-2 2 7 resident proteins such as PST02549 or TaEDC4. However, we observed this phenomenon 2 2 8 only when the two proteins co-accumulated, indicating that both are required to increase P- body size. The biological significance of the association between PST02549 and TaEDC4 as 2 3 0 well as the increase in P-body size remain to be further investigated in wheat. The pipeline we used in this study allowed us to retrieve informative data for more 2 3 2 than 50% of the candidate effectors we tested. We recently obtained informative data for 40% pathogens, regardless of their host plant.

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We identified plant interactors of candidate effectors, some of which may represent leucine rich repeat (NB-LRR, also referred to as NLR) proteins to become 'sensor domains'  domain of EDC4 (Table S2). Therefore, our predicted host targets can be a valuable source 2 4 6 of new 'baits' for engineering NLR genes with sensor domains. from Tribe 54, as well as PST18220 and PST18221 from Tribe 238, were both retained due to  cDNA isolated from wheat leaves 14 days after inoculation with a virulent isolate of P.  Table S1). Truncated versions of PST15391 and PST18447 were obtained by PCR 2 7 2 cloning. All PCR-generated DNA fragments were verified by sequencing after cloning into confocal microscopy assays. two days after infiltration for further protein isolation or microscopy. localisation pattern in at least three independent observations. Image analysis was performed the 'measure' tool of Fiji was used to measure manually-drawn lines matching the apparent this project. Categorical scatterplots were generated with R, using the ggplot2 package and 2 9 7 an in-house developed script (Text S1). Frozen leaves were ground to a powder using a mortar and pestle. Total proteins 3 0 2 were extracted as previously described (Petre et al., 2015a). Ten microliters of isolated 3 0 3 protein was separated on a 15% SDS-PAGE gel, and the protein content was estimated by 3 0 4 Coomassie Brilliant Blue (CBB) staining. Immunoblot analysis was performed as previously Cruz Biotechnology), rat anti-RFP 5F8 antibody (Chromotek, Munich, Germany) and a HRP- conjugated anti-rat antibody.  Table   3 2 1 S2). We thank the Norwich Rust Group for discussions, Dr. Vanessa Segovia (Norwich, is supported by the Gatsby Charitable Foundation and the BBSRC. were retained. The full dataset used to draw these figures is shown in Table S2. The 16 candidate effectors used in this study are shown in the middle column. Colours Triticum aestivum L. using the BLASTp algorithm.