Title : Multiplexed effector screening for recognition by endogenous resistance genes using 2 positive defense reporters in wheat protoplasts 3 4

Abstract

(Jensen and Saunders, 2023). We only expressed AvrSr50 from a plasmid. We also included the 1 0 3 controls, the reporter alone and the reporter with Avr or R alone (Zenodo Supplemental Data S1).

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We measured luminescence at 3, 4, 5, 6, and 18 h post-transfection (hpt). We observed a gradual strongest at 18 hpt in all treatment groups except for the ones expressing Avr/R pairs. In cv. interaction treatment groups ( Figure 1A and B). The lack of continuous increase of luciferase 1 1 3 activity specifically in protoplast expressing Avr/R pairs suggests that their co-expressing leads 1 1 4 to cell death in wheat protoplasts soon after transfection. We concluded that 4 hpt is a suitable time point to capture Avr/R-dependent transcriptional 1 1 7 changes before strong HR induction based on our time course analysis. We repeated the Illumina platform generating >20 million 150 bp paired-end reads per replicate. We used kallisto Avr and R genes to investigate their specific expression pattern at 4 hpt. This analysis clearly showed that all genes are correctly expressed and at similar levels, including in treatment groups 1 2 6 that co-express Avr/R gene pairs (Figure 2 A). In addition, it revealed that the endogenous 1 2 7 expression of Sr50 in cv. GaboSr50 is much lower level than the overexpressed of Sr35 in cv.
Fielder from a plasmid under the UBI promoter. Next, we performed principal component  these results suggest that these data could be used to identify reporter genes specifically to cellular components of cell periphery (GO:0071944) and plasma membrane (GO:0005886) in Supplemental Data S4). We found molecular functions GO terms being enriched for calcium ion confirms that Avr/R expressing protoplasts undergo defense signaling before the onset of strong 1 5 7 cell death. We were then interested in genes that are commonly upregulated by the Avr/R interaction in In summary, we observed the upregulation of genes involved in defense signaling in wheat protoplasts expressing Avr/R pairs. The promoters of commonly upregulated genes, by two 1 6 7 Avr/pairs and in two wheat cultivars ( Figure 2F), are promising candidates to generate Avr/R 1 6 8 inducible reporters. We further investigated the expression patterns of the 66 commonly upregulated genes to select a 1 7 1 subset whose promoters we could use as positive defense signaling reporters. We selected 13 promoters, consisting of 600-800 bp upstream of their respective genes' start codons. We To test the activity of our thirteen candidate defense promoters, we generated promoter fusion 1 8 2 constructs driving the expression of green-emitting luciferase (E-Luc). These plasmids are -7, 10-12, 14-16]), respectively. We co-transfected each of these plasmids Triticum monococcum (McIntosh et al., 1984). These results indicate that our new ratiometric 1 9 2 reporter system is able to detect specific defense activation downstream of two Avr/R 1 9 3 interactions when the R gene is expressed endogenously.

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We next addressed whether the induction of D2, D14, and D15 promoters is specific to 1 9 5 AvrSr50/Sr50 and AvrSr35/Sr35 interactions. We extended our analysis to the recently published pD15 can serve as reporters of Avr/R-mediated response more broadly. We used protoplasts with a plasmid that expresses AvrSr27 under the control of the UBI promoter. We compared the reporters in wheat independent of the wheat cultivar and the specific Avr/R combination used. In addition, this suggests that our protoplast assay is applicable to testing wheat cultivars that carry 2 0 7 endogenous R genes and does not require overexpression of a cloned R gene. We aimed to illustrate the broad applicability of our new assay in a wide range of wheat cultivars The cultivars Avocet (Yr8) and Fielder were tested with AvrSr27/Sr27 and AvrSr35/Sr35 combinations, respectively. We include a negative control in all cultivars by expressing the R 2 2 0 gene together with an un-recognized Avr (Figure 4). In all cultivars we tested, a consistent robust Avr/R-pair compared to the negative control. The relative normalized luminescence and its wheat cultivars tested.

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The positive defense reporter assay enables multiplexed screening of Avr candidates in bulk 2 2 7 To explore multiplexed screening of Avr candidates using our protoplast assay, we transfected a 2 2 8 pool of ten Avr candidates, including AvrSr50, AvrSr27, AvrSr35, and seven other wheat rust 2 2 9 candidate effector genes used in our studies, into protoplasts isolated from cv. GaboSr50 and cv. difference between the two treatment groups ( Figure 5A). In contrast, there was a clear Avr- unspecific reduction in red luminescence induced by both multiplexed mixtures in both cv. Gabo 2 4 0 and cv. GaboSr50 protoplast ( Figure 5B). This indicates that the ratiometric dual-color 2 4 1 luciferase system combined with pDefense reporter is highly sensitive in detecting Avr/R 2 4 2 interactions and more specific than general luciferase based cell death based assays. Overall, 2 4 3 these results showed that our positive defense reporter protoplast assay can be used to screen a 2 4 4 pool of Avr candidates against a wheat cultivar endogenously expressing the recognizing R gene. Here, we describe an improved wheat protoplast assay to screen avirulence effector candidates 2 4 7 and study cellular defense signaling in wheat. We identify Avr/R-induced promoters and use 2 4 8 them to generate reporter constructs that produce a positive readout during Avr/R interaction. In addition, we also reduce variability of luminescence measurement by adapting a ratiometric expressing R genes endogenously.

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This approach has several advantages over previous methods applied to identify Avr/R 2 5 6 interactions. Firstly, it does not require a cloned and overexpressed R gene to test the interaction.

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We demonstrate that the positive defense reporter assay is suitable to test interactions directly in resources. Lastly, our new wheat protoplast reporter assay enables us to study a wide range of 2 6 6 plant defense signaling directly in wheat. This is highlighted by our RNAseq analysis of defense 2 6 7 signaling in wheat protoplasts. Our results show that genes responsible for calcium ion binding, Interestingly, one of our marker genes (the gene of promoter D14) encodes a predicted wheat effector ToxA was shown to interact with a wheat NHL protein, TaNHL10, to promote cell death will benefit from our defense specific promoters, in particular D14, to investigate downstream 2 9 4 defense signaling components in wheat.

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We acknowledge future improvement will further extend the applicability of our positive defense 2 9 6 reporter as described here. Similar to the previous wheat protoplast cell-death based assays (Saur al., 2023) will allow us to reduce required plasmid amounts. Our study is complementary to a 3 0 7 recent report that describes highly multiplexed avirulence effector recognition screening using of five Sr genes including AvrSr13/Sr13 and AvrSr22/Sr22 but not for Sr21, Sr26, and Sr61.

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Such large scale avirulence effector screening approaches will be much more efficient when 3 1 6 combined with our positive reporter assays that will allow researchers to first identify avirulence 3 1 7 effector pools that are recognized by a specific wheat cultivar. In addition, further improvements 3 1 8 of the reported negative enrichment screen will likely enable screening in wheat cultivars 3 1 9 expressing R genes endogenously similar to our assay. Hence the combination of both and GaboSr50 were grown for 7-9 days in a growth cabinet with 150 µmol/m 2 /s light intensity CaCl 2 (10 mM). BSA 0.1% w/v). The dish was wrapped in aluminum foil to shield it from light 3 3 4 and placed on an orbital shaker, horizontally rotating at 60 RPM, for three hours. Protoplasts protoplasts/mL using MMG (MES-pH5.7 (4 mM), Mannitol (0.4 mM), MgCl 2 (15 mM)).

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Lower quality plasmid DNA (e.g. from miniprep kits) typically resulted in low transfection rates.