A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

Background The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection. Results We developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method. Conclusions We have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

Examples of environmental factors that can lead to UPR include temperature changes, ionic and to their denaturation and accumulation in different organelles, including the ER, leading to stress. 53 In plants, there are at least two different mechanisms by which ER stress can be perceived and 54 activate a signaling cascade that triggers UPR. In the Inositol-requiring enzyme 1 (IRE1) 55 pathway, luminal binding proteins (BiPs) interact with the ER-membrane protein IRE1 in the ER 56 lumen. When unfolded proteins accumulate, they are bound by BiPs, releasing IRE1 proteins that 57 then form dimers which unconventionally splice basic leucin zipper (bZip) 60 mRNAs in the 58 cytosol. The spliced mRNA translates into a functional transcription factor that shuttles to the 59 nucleus and promotes the upregulation of genes that contain UPR responsive elements (UPREs) 60 and ER stress elements (ERSEs) in their regulatory regions (Hayashi et  found to interact with FKBP15-2, a plant peptidyl-prolyl cis-trans isomerase which was found to 83 be required for ER stress mediated immunity (Fan et al., 2018). However, the lack of a method 84 for screening proteins that interfere with plant UPR has made it difficult to identify effectors in 85 other pathogens that might play a role in this process. 86 Though the conserved pathways of UPR signaling in plants have been  mechanism is yet to be reported.

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Here we report a method for screening proteins, and potentially other molecules or conditions, 101 that influence plant UPR. This method relies on fluorescence measurements of Nicotiana 102 benthamiana leaf discs transiently expressing two genetic constructs. One of them expresses the 103 protein of interest, while the second plasmid encodes an ER-stress responsive promoter 104 controlling the expression of enhanced yellow fluorescent protein (eYFP). By using a subset of 105 proteins from a library of secreted proteins (i.e. putative effectors) from the maize pathogen 106 Ustilago maydis, we were able to identify one protein that inhibits UPR signaling in plants. After 107 validation by more classical, laborious methods, this simple approach allows for the screening 108 and identification of new players in plant UPR that may have a role in specific conditions.

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A fluorescence-based assay to measure UPR stress 113 We developed a method that measures relative UPR stress and signaling in plants (Fig. 1). By 114 co-expressing a reporter construct and a protein of interest, interference in UPR signaling can be 115 assessed and new players in this cellular mechanism can be identified. Additionally, the same 116 reporter plasmid could be used to assess the influence of other molecules or environmental 117 conditions on UPR signaling.  To establish the assay presented in Fig. 1, several conditions were tested and optimized to 140 guarantee the reliability of the assay. First, a suitable UPR responsive promoter had to be 141 identified which shows sufficient strength and high reproducibility in its response to UPR stress. 142 We cloned the promoter regions from four genes that had been reported to be upregulated in ER days later, we infiltrated the same leaves with 5 μg/mL Tm to induce UPR and measured eYFP 148 levels approximately 24 hours after the second infiltration ( Fig. 2A). The regulatory region of 149 SKP1 was the only one that did not lead to a significant increase in eYFP fluorescence after UPR 150 induction. From the remaining promoters, bZIP60 showed the highest fold change of eYFP 151 expression under ER stress conditions (6.03 ± 2.41), followed by BIP1 (5.57 ± 2.19), and BIP3 152 (4.27 ± 3.51). Considering the high variability observed for pBIP3 and the low fluorescence 153 levels in samples with the bZIP60 promoter, we concluded that pBIP1::eYFP was the most 154 suitable construct for this method. Therefore, all remaining optimization steps were performed 155 using pBIP1::eYFP as the reporter construct.

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The second factor we optimized was the measurement time after UPR induction. We tested 157 samples at 6, 12, 24, and 48 hours after 5 μg/mL Tm infiltration and compared them to the mock 158 treated samples (Fig. 2B). The timeseries shows a gradual increase in eYFP fluorescence after   After determining that the regulatory region of BIP1 displayed a good signal to noise ratio 167 after 24 h of ER stress, we determined the optimal Tm concentration to induce promoter activity. plasmids (OD600 nm = 0.2) resulted in a 4.28 ± 1.52 fold change, while a 2:1 ratio of pBIP1::eYFP 178 to p35S::mCh-P2A-mCh (OD600 nm = 0.2 and 0.1, respectively) led to a 5.47 ± 1.27 fluorescence 179 increase. Importantly, samples in which the reporter plasmid had a lower OD600 nm relative to the 180 expression plasmid had significantly higher mCh fluorescence (Fig. 2E). Thus, a 1:2 ratio of 181 pBIP1::eYFP to p35S::mCh-P2A-mCh (OD600 nm = 0.1 and 0.2, respectively ) leads to similar 182 eYFP induction, while allowing for higher expression of candidate genes. It is also important to 183 note that eYFP induction upon UPR was lower in this assay when compared to the previous 184 experiments. This is likely due to competition in the transient production of two proteins as 185 opposed to one. Nonetheless, in these conditions, eYFP is more than four times more abundant in 186 ER stressed plant leaves. that the conditions we optimized for our fluorescence-based method leads to ER stress. 199 Finally, we tested whether our conditions can detect UPR interference using proteins known to 200 be involved in UPR signaling. We co-infiltrated the pBIP1::eYFP reporter construct with either: 201 p35S::mCh-P2A-mCh, as a reference for unaltered UPR signaling; p35S::IRE1a (AT2G17520), 202 which leads to the upregulation of UPR-related genes; or p35S::HY5 (AT5G11260), which is  proteins from a library of putative effectors from the biotrophic fungal pathogen U. maydis to test 216 whether our method could link any of them to UPR signaling (Fig. 4). In both mock and Tm 217 treated samples, we observed relatively high eYFP fluorescence variation between samples. We 218 therefore decided to apply a strict significance threshold of p ≤ 0.01 in our ANOVA tests. In repetitions in Tm-treated samples was also observed (Fig. 5B). In trying to understand the source 232 of this variation, we considered whether it could be due to changes in protein expression between 233 the two replicates. Because the plasmids encoding the candidate genes also express mCh in 234 equimolar amounts, we used this protein's fluorescence as an estimate for protein levels of the 235 different constructs (Fig. 5C). We found that there was indeed variation in protein levels between 236 the two replicates in some samples and this is a factor that should be considered when using this 237 method. Nonetheless, the putative effector UMAG_0592724-370 consistently downregulated pBIP1 238 activity to approximately half of what was measured in the mCh control sample ( Fig. 5A and B).

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In Tm infiltrated leaves, qRT-PCR analysis of the same maker genes measured in Fig. 3A 240 showed that expression of UMAG_0592724-370 led to a significant decrease in CNX1, SKP1, and 241 PR1 expression, but not bZIP60 (Fig. 5D). This indicates that UMAG_0592724-370 can interfere 242 with UPR, either downstream of bZIP60 or in a signaling pathway-specific manner.

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There was one more observation we noted that might influence some of the variability of the   The most commonly used method to link proteins of interest with UPR is qRT-PCR for ER  Additionally, inconsistencies in protein expression between samples, as seen in Fig. 5C, E, and F, 306 and known phenotypic changes that occur between transient and stable protein expression have to 307 be taken into account when analyzing data obtained by this method (Bashandy et al., 2015).

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Because of this, we recommend that an initial screen be used to short list proteins for a second 309 round of testing. Proteins that show a consistent effect on eYFP expression across the two 310 replicates can then be validated by qRT-PCR and further characterized. genes with that expression profile, we tested the regulatory region of 4 of them: SKP1, bZIP60, 315 BIP1, and BIP3 (Fig 2A). In the case of SKP1, Fig. 3A shows that this gene is only moderately 316 upregulated after Tm infiltration. It was therefore not surprising that we could not detect its 317 upregulation in the fluorescence-based assay. This highlights a disadvantage of this method, 318 namely that it is limited to the discovery of proteins with a strong influence on UPR signaling.

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BiP proteins are essential for UPR and their expression is tightly regulated during this process. 320 bZip60, on the other hand, has a role in early ER stress signaling events and its mRNA is 321 transcribed in non-stress conditions so that it can be unconventionally spliced during UPR (Iwata  The co-expression of the known UPR inducer IRE1a or inhibitor HY5 with our reporter 350 construct showed the expected correlation with eYFP expression following induction of UPR.

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Together with the measurement of UPR marker genes by qPCR, Fig. 3 shows that the optimal 352 conditions determined in Fig. 2 effectively lead to UPR and that the method is suitable for 353 discovering new proteins that influence this mechanism.

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Our small screen with a set of U. maydis effectors (Fig. 4) led to the identification of a protein, 355 UMAG_0592724-370, which seems to interfere with this process. This effector consistently led to 356 the down regulation of eYFP expression from the reporter construct ( Fig. 5A and B) and 3 out of 357 the 4 measured UPR marker genes (Fig. 5D). It is worth noting that the expression of 358 UMAG_0592724-370 did not influence bZIP60 transcription, which is commonly upregulated upon 359 ER stress. It did however strongly downregulate pathogenesis related 1 (PR1) expression, which 360 is widely reported to be upregulated upon SA signaling (Seyfferth & Tsuda, 2014). It is tempting 361 to speculate that the influence of UMAG_0592724-370 on UPR may be dependent on SA signaling, 362 rather than a more generic UPR inhibition. However. further functional characterization of this 363 protein is needed to better understand its role in UPR interference and pathogenesis. Nonetheless,   Our method enables the testing of gene, and potentially small molecule, libraries using 375 relatively limited resources and time. By using fluorescence as the output of the assay, which can 376 be measured from leaf discs in 96 well plates, many factors can be easily tested in parallel. In 377 fact, our pilot experiment tested 35 proteins and identified one which influences UPR signaling. 378 We anticipate that this reporter system will lead to the discovery of new players in plant UPR 379 signaling, particularly those involved in biotic interactions or that play a role in specific 380 environmental conditions. This will lead to a better understanding of this ubiquitous and very 381 complex cellular homeostasis mechanism and its role in plant biology.     Table 1. Whenever necessary, BsaI 411 restriction sites native to the coding sequences of the promoters or putative effectors were 412 mutated. Silent mutations were introduced by site directed mutagenesis (Liu & Naismith, 2008) 413 to preserve the native amino acid sequence and maintain the efficiency of the Golden Gate 414 cloning method (Engler et al., 2008). In the case of the fluorophores, eYFP was re-cloned from a 415 different vector system using primers with adaptors to enable its compatibility with our cloning 416 strategy (Table 1) Table 1.   Acknowledgements 503 We would like to acknowledge the GMI/IMBA/IMP service facilities, particularly the 504 molecular biology services for Sanger sequencing and support when using the plate reader. We   Table 2). Detailed information on the 517 remaining plasmids is available upon request.