New Results

PAT mRNA decapping factors function specifically and redundantly during development in Arabidopsis

Zhangli Zuo, Milena Edna Roux, Yasin F. Dagdas, Eleazar Rodriguez, Morten Petersen
doi: https://doi.org/10.1101/2022.07.06.498930

Abstract

Evolutionarily conserved PAT1 proteins activate mRNA decay through binding mRNA and recruiting decapping enzymes and other factors hence optimize transcriptional reprogramming during development. Here, we generated multiple mutants of pat1 (Protein Associated with Topoisomerase II), path1 and path2 and inspected their growth and leaf morphology phenotype. pat triple mutants exhibit extreme stunted growth and all mutants with pat1 exhibit leaf serration while mutants with pat1 and path1 all display short petioles. All 3 PATs can be found localized to Prossessing Bodies (PBs) upon auxin treatment and RNA-seq analysis indicate that all 3 PATs redundantly regulate auxin responses. Moreover, shade avoidance and NAC genes are misregulated in pat1path1 double and pat triple mutants suggesting PAT1 and PATH1 function in petiole elongation and leaf patterning. In conclusion, PAT proteins exhibit both specific and overlapping functions during different stages of plant growth and our observations underpin the importance of the mRNA decay machinery for proper development.

Introduction

The RNA-binding proteins (RBPs) PAT1(Protein Associated with Topoisomerase II) family proteins are highly conserved through eukaryotes and mediate mRNA decay in cytoplasm. As a central platform, PAT1 forms a heterooctameric complex with LSM(Like-sm)1-7 at 3’ end of oligoadenylated mRNAs to engage transcripts containing deadenylated tails thereafter recruits decapping enzymes and other factors for 5’-3’ decay, these decapping complex and mRNAs can diffuse in cytoplast or aggregate into distinct cytoplasmic foci called PBs (Brengues et al., 2005; Balagopal and Parker, 2009;Ozgur et al., 2010; Chowdhury et al., 2014; Charenton et al., 2017; Lobel et al., 2019). The deadenylated mRNAs could also be degraded 3’-5’ via exosomal exonucleases and SUPPRESSOR OF VCS (SOV)/DIS3L2. mRNA decay regulates transcriptome shift and thereby impacts organism development and responses to stresses (Newman et al., 2017; Essig et al., 2018; Xu and Chua, 2012; Merret et al., 2013; Roux et al., 2015; Perea-Resa et al., 2016).

Yeast, fungi and green algae encode one PAT1 paralog respectively and vertebrates encode two while most plants possess multiple PAT1 family members (Zhang et al., 2020). For example, moss has two PAT1 paralogues, rice holds four and Arabidopsis encodes three, named PAT1, PATH1 and PATH2 (Zhang et al., 2020; Roux et al., 2015). Deletion of well conserved PAT1 gene exhibits temperature sensitive phenotype in yeast and embryonic lethality in Drosophila melanogaster respectively (Wyers et al., 2000; Pradhan et al., 2012). Besides, we previously reported that Arabidopsis pat1 mutant exhibit auto-immune phenotype, PAT1 (AtPAT1) localizes to PBs and is phosphorylated on serine 208 by MAP kinase 4 (MPK4) in response to bacterial flagellin (Roux et al., 2015). Loss-of-function of AtPAT1 inappropriately triggers the immune receptor SUMM2, and Atpat1 mutants consequently exhibit dwarfism and de-repressed immunity (Petersen et al., 2000, Roux et al., 2015, Zhang et al., 2012). Thus, PAT1 is under immune surveillance and PAT1 function(s) are best studied in SUMM2 loss-of-function backgrounds (Roux et al., 2015).

In addition to PAT1, Arabidopsis encodes 2 other PATs, PATH1 and PATH2. So far, little is known about the functions of these 3 PATs during plant development. Here we have disrupted the mRNA decapping components PAT1, PATH1 and PATH2 in the summ2 background (Roux et al., 2015) which allows us to study this process without autoimmunity interference. We have revealed all three PAT proteins interact with the LSM1 decapping factor and localize to PBs upon different stimuli perception. Through observing developmental phenotype of multi-pats mutants and performing RNA-seq to examine differently expressed genes we identified PAT1 is mainly responsible to keep regular leaf pattern and work redundantly with other 2 PATs during plant development.

Results

PAT mRNA decapping factors interact with LSM1

Arabidopsis genome encodes 3 PAT mRNA decapping factors: PAT1, PATH1 and PATH2 (Roux et al., 2015). We previously reported that PAT1 localizes to PBs and can be found in complexes together with the decapping component LSM1 (Roux et al., 2015). To test if PATH1 and PATH2 are also in mRNA decay complex, we transiently expressed all three PAT-HA fusions with YFP-LSM1 and performed coimmunoprecipitation assays. All three PAT-HA fusions could be detected in LSM1 immunoprecipitates (Fig 1A), indicating that, similar to PAT1, PATH1 and PATH2 also localize to LSM1 positive compartments. In line with this, a previous study looking for LSM1 associated proteins detected all three PATs proteins in LSM1 pull downs (Golisz et al., 2013).

Fig 1.
Fig 1. PATH1 and PATH2 are also mRNA decapping factors.

(A) Co-IP between the three PAT-HA and YFP-LSM1 fusions. Proteins were transiently co-expressed in N. benthamiana and tissue harvested 3 days post-infiltration. Immunoblots of inputs (left panels) and GFP IPs (right panels) were probed with anti-HA antibodies (upper pannel) and anti-GFP antibodies (middle panel for YFP-GUS and bottom panel for YFP-LSM1). (B) 6-week-old plants of summ2-8 and multiple pat mutants grown in soil in a chamber with 8/16hrs light/dark at 21°C. A representative plant for each line is shown. The scale bar indicates 1cm.

PAT(s) loss-of-function mutants exhibit different growth phenotypes

While we had detected PAT1 in PBs and found it under SUMM2 surveillance (Roux et al., 2015), little was known about mRNA targets or functions of the three PATs in Arabidopsis. To address this, we generated single, double and triple knockouts (KOs) of the three PATs in the summ2-8 background to avoid immune activation. path1 and path2 mutants were therefore generated in pat1-1summ2-8 and summ2-8 by CRISPR/CAS9-mediated genome editing (Fig 1B&S1A). Fig S1B shows the sequences of PATH1 and PATH2 in 2 independent pat triple mutants. The growth phenotypes of 6 weeks-old soil-grown plants of pat single, double and triple mutants at 21 °C with 8/16 hrs light/dark photoperiod are shown in Fig. 1B& S1A. Both independent lines of pat1-1path1-4path2-1summ2-8 and pat1-1path1-5path2-2summ2-8 exhibited markedly stunted growth compared to the other pat single or double mutants (Fig. 1B, S1B) indicating functional redundancy of PATs in regulating plant development. path1-4summ2-8 and path2-1summ2-8 have leaf number and shape similar to summ2-8 while pat1-1summ2-8, pat1-1path1-4summ2-8, pat1-1path2-1summ2-8 and pat1-1path1-4path2-1summ2-8 grow fewer leaves and with serration shape indicating PAT1 has specific function in regulating leaf serration (Fig. 1B). Furthermore, complementation lines of pat1-1path1-4summ2-8 and pat1-1path2-1summ2-8 with Venus-PATH1 and Venus-PATH2 respectively show the same growth phenotype as pat1-1summ2-8 (Fig. S1C&D).

PATs localize to PBs upon different stimuli perception

mRNA decapping complex and mRNAs can aggregate into PBs in the cytoplasm (Ozgur et al., 2010). To investigate if all three PAT proteins may also localize into PBs, we exposed seedlings expressing Venus-fusions of the PAT proteins (Fig. S1C-F) to the phytohormones auxin (IAA), cytokinin, ethylene precursor ACC, bacterial flagellin peptide flg22 and extracellular ATP. All stimuli induced massive increase of Venus-PAT1 distinct foci in roots within 20 min (Fig. 2). PATH1 only localized into PBs in response to IAA while PATH2 also enters PBs under ACC treatment (Fig 2). These observations indicate PAT1 plays a main role in forming PBs compared to PATH1 and PATH2.

Fig 2.
Fig 2. PATs relocalize to PBs upon stimuli perception.

Venus-PATs expressing seedlings in MS growth medium or 20min after treatment with MS containing 0.5µM IAA, 1µM Cytokinin, 25µM ACC, 5µM flg22 or 0.1mM ATP. Representative images are shown. Scale bars indicate 10µm.

All three PATs redundantly regulate plant development

To better quantify growth phenotype of these pats mutants, we dissected 6-week-old plants of Col-0 and multiple pat mutants (Fig 3A) and recorded leaf (leaf blade length >1mm) numbers. All mutants except path1-4summ2-8 and path2-1summ2-8 developed fewer leaves than Col-0 and summ2-8, while pat1-1path1-4path2-1summ2-8 exhibit least leaf numbers (Fig 3B), indicating redundancy of all 3 PATs in regulating plant growth.

Fig 3.
Fig 3. Leaf morphology of pat mutants.

Leaf development (A), leaf numbers (B), leaf blade length (C), leaf petiole length(D), leaf serration numbers(E) and serration levels (F) of 6 week-old plants of Col-0, summ2-8, pat1-1summ2-8, path1-4summ2-8, path2-1summ2-8, pat1-1path1-4summ2-8, pat1-1path2-1summ2-8, path1-4path2-1summ2-8 and pat1-1summ2-8, path1-4summ2-8, path2-1summ2-8, pat1-1path1-4summ2-8, pat1-1path2-1summ2-8 and pat1-1path1-4path2-1summ2-8. The scale bar indicates 1cm. Leaf-serration levels are expressed as the distance from tip-to-midvein divided by the distance from sinus-to-midvein for different teeth.

PATs play different roles in regulating leaf morphology

Leaf morphology is an essential part of phytomorphology. We therefor inspected leaf size and shape of the latest expanded leaves from the multiple pat mutants. Similar to leaf number, pat1-1path1-4path2-1summ2-8 exhibit shortest leaf blade and all mutants except path1-4summ2-8 and path2-1summ2-8 developed shorter leaf blade than Col-0 and summ2-8 (Fig 3C). Petioles connect leaf lamina to plant stem, pat1-1summ2-8 exhibit shorter petioles than Col-0 and summ2-8, while pat1-1path1-4summ2-8 and pat1-1path1-4path2-1summ2-8 showed shortest petioles (Fig 3D), indicating only PAT1 and PATH1 are regulating leaf petiole length and PAT1 plays the main role.

Evolutionarily, variation in plant leaf shape reflects natural selection operating on function. To better understand PATs function in leaf serration, we counted serration numbers per half leaf and measured serration level on the latest expanded leaves from the multiple pat mutants (van Wijk et al., 2018). Interestingly, pat1-1summ2-8 displayed increased number of serrations compared to control plant and pat1-1path1-4summ2-8 but not pat1-1path2-1summ2-8 showed the most serrations (Fig 3E), suggesting both PAT1 and PATH1 but not PATH2 function in regulating leaf serration numbers. All mutants with pat1 loss-of-function exhibit higher serration level than other mutant combinations and pat1-1path2-1summ2-8 displayed lowest serration level in plants with PAT1 mutated (Fig 3F), indicating PAT1 is regulating leaf serration level, and PATH2 may play opposite role. All these results suggest that the function of PATH1 and PATH2 are redundant to the other PATs, while PAT1 serves a main and specific function in development.

PAT(s) loss-of-function mutants exhibit different transcriptome

Plant and organ growth is tightly regulated by their developmental program. To identify genes which affect different developmental programs regulated by mRNA decapping factor PATs, we performed RNA-seq from plants of pat1-1path1-4path2-1summ2-8 and all single and double mutant combinations shown in Fig 1B. Principal Component Analysis (PCA) Plot (Fig. 4A) exhibit general similarities of these 8 lines, revealing that mutants with pat1 are different from the other plants based on PC1, and based on PC3, pat1-1summ2-8 and pat1-1path1-4summ2-8 are most similar to each other and pat1-1path1-4path2-1summ2-8 positions in between pat1-1path2-1summ2-8 and pat1-1path1-4summ2-8. Data File S1 exhibit differently expressed genes in multiple pat mutants which are clustered in Fig. 4B, and numbers of shared DE (differently expressed) genes are summed up in Fig. 4C. GO term analysis on misregulated genes in pat1-1path1-4path2-1summ2-8 showed that: i. genes involved in oxidation-reduction are largely mis-regulated, ii. transcripts involved in oxidative stress response accumulated, and iii. transcripts responsible for auxin response and signaling and growth regulation are reduced in pat triple mutant (Fig. S2) (Huang et al., 2009).

Fig 4.
Fig 4. RNA-seq analysis on pat mutants.

(A) 3-D PCA plot for general similarity of pats single, double and triple mutants with bio-triplicates. (B) Heat map clustering of differentially expressed genes for comparison of pat mutants and summ2-8. Pearson’s metrics were used in hierarchical clustering of the genes. In the plot, red indicates high expression and blue low expression. (C) Numbers of shared differently expressed genes in pat mutants.

PATs regulate different genes expression during development

Leaf initiation and outgrowth have been reported under auxin regulation (Xiong, Y.&Jiao, Y, 2019). We therefore took closer look at the auxin responsive genes including SAURs (SMALL AUXIN UP RNAs), IAAs, MYBs (MYB DOMAIN PROTEINs), WAG1, WAG2 etc. They were least expressed in pat triple mutant compared to all pat double mutants and pat1 single mutant in which the auxin responsive genes were also down regulated compared to summ2-8 and path1 and path2 single mutant (Fig. 5).

Fig 5.
Fig 5. PATs regulate different genes expression.

Heat map clustering of differentially expressed genes including auxin responsive genes, shade avoidance related genes and leaf serration related genes.

Light mediated petiole elongation during shade avoidance involves cell wall modification by XTHs (Xyloglucan Endotransglucosylase/Hydrolases) and is enhanced by PIFs (PHYTOCHROME-INTERACTING FACTORS) (Sasidharan, R., 2020; Xie, Y., 2017). XTH7 and PIL2/PIF6 were downregulated in pat1-1summ2-8, pat1-1path1-4summ2-8 and pat triple mutants which exhibit shorter petioles than all other mutants (Fig.3& 5), suggesting PAT1 and PATH1 may regulate XTH7 and PIL2 to control petiole elongation.

The NAC family member CUC2 (CUP SHAPED COTYLEDON 2) and PIN1 auxin efflux carrier have been modeled to explain leaf serration and CUC3 has also been reported to be essential to keep serration (Bilsborough G.D., 2011; Serra L., 2020), however CUC2 and CUC3 were downregulated in the serrated mutant pat1-1summ2-8 while we found other 2 NAC family gene ANAC003 and ANAC016 specifically accumulated in all serrated mutants indicating PAT1 and PATH1 may regulate other NAC genes expression for leaf patterning (Fig. 5). Nevertheless, all these results indicate all 3 PATs regulate different genes expression during leaf development.

Discussion

Plant development requires massive overhauls of gene expression (Miyamoto et al., 2015). Our phenotypic characterizations showed that, compared to pat1-1summ2-8, leaf serration and dwarfism are more severe in pat1-1path1-4summ2-8, while pat1-1path2-1summ2-8 exhibits less serration and normal petioles, indicating that PAT1 and PATH1 share some functions in leaf serration and petiole elongation while PATH2 may have opposite functions (Fig 3). However, loss-of-function of all 3 PATs causes severe dwarfism compared to any single or double mutants; together with this, pat triple mutant accumulates the most differently regulated genes of which 348 genes specifically misregulated in pat triple mutants (Fig 4C), indicating some redundancy and subfunctionaliztion among the three PATs in development (Duarte et al., 2006). The fact that all three PATs localized into PBs in response to IAA treatment also confirms functional redundancy of the PAT proteins (Fig. 2). While shared DE genes analysis manifests more DE genes shared by pat1-1path1-4summ2-8 and pat triple mutant than the other two pat double mutants and pat triple mutant, probably suggesting mutation of PAT1 and PATH1 provide the highest contribution to the developmental phenotypes of pat triple mutant. Interestingly, pat1-1summ2-8 mutants still exhibit dwarfism and leaf-serration, while neither path1summ2-8 nor path2summ2-8 mutants exhibit developmental defects (Fig1& 3). Our RNA-seq data shows that 382 transcripts are differentially expressed in pat1-1summ2-8 compared to only 54 genes in path1-4summ2-8 and 279 in path2-1summ2-8 (Data File S1). While we do not know how these differences contribute to the developmental phenotype of pat1, these observations suggest a non-redundant function of PAT1 during plant development. In line with this, we previously reported that PAT1 specifically functions in response to osmotic stress (Zuo et.al., 2021) while all 3 PAT proteins function redundantly during Turnip mosaic virus infection (Zuo et.al., 2022) and other development processes including apical hook and lateral root formation (not published yet).

Besides pat tripe mutants, other mRNA decapping deficient mutants also exhibit abnormal development or postembryonic lethality (Xu et al., 2006; Xu and Chua, 2009; Perea-Resa et al., 2012; Zuo et al., 2022b). The stunted growth phenotype and downregulation of developmental and auxin responsive mRNAs in the pat triple mutant (Fig. 5&S2) may support a model in which defective clearance of suppressors of development hampers developmental programming. Several studies also implicated yeast PAT1-LSM1-7 complex inhibit exosome mediated 3’-5’ decay leading to certain mRNA stabilization and accumulation (Tharun, 2009; Gatica et al., 2019). Therefore, Arabidopsis pats loss-of-function mutants may also lose protection of some transcripts from exosome mediated decay, these destabilized transcripts could be auxin responsive genes and growth-related genes.

In conclusion, we have shown that PAT1, PATH1 and PATH2 interact with the LSM1 decapping factor and localize to PBs upon different stimuli perception (Fig. 1A &2). Through observing developmental phenotype of multi-pat mutants, investigating cellular localization of PAT proteins and performing RNA-seq to examine differently expressed genes we identified PAT1 serve as the main role during development especially in leaf morphology and work redundantly with other 2 PATs during plant development.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was used as a control. All mutants used in this study are listed in Table S1. T-DNA insertion lines for At1g79090 (PAT1) pat1-1 (Salk_040660), At1g12280 (SUMM2) summ2-8 (SAIL_1152A06) and double mutant of PAT1 and SUMM2 pat1-1summ2-8 have been described (Petersen et al., 2000; Zhang et al., 2012; Roux et al., 2015). pat1-1path1summ2-8 and path2summ2-8 mutants were generated using CRISPR/CAS9 following standard procedures with plasmid pHEE401 containing an egg cell-specific promoter to control CRISPR/CAS9 (Wang et al., 2015). Independent T1 pat1-1path1hetsumm2-8 and path2hetsumm2-8 plants were used in crosses to produce pat1-1hetpath1 hetpath2 hetsumm2-8, Cas9 free mutants. Homozygous F2 plants were selected and used for experiments.

Plants were grown in 9×9cm or 4×5cm pots at 21°C with 8/16hrs light/dark regime, or on plates containing Murashige–Skoog (MS) salts medium (Duchefa), 1% sucrose and 1% agar with 16/8hr light/dark.

Plant treatment and confocal microscopy

Seedlings were grown in liquid MS medium for 5 days, confocal microscopy pictures were taken with a Leica SP5 inverted microscope after 20min treatment with MS containing 0.5µM IAA, 1µM Cytokinin, 25µM ACC, 5µM flg22 or 0.1mM ATP.

RNA-seq analysis

Total RNA was extracted from 6-week-old soil grown plants including summ2-8, pat1-1summ2-8, path1-4summ2-8, path2-1summ2-8, pat1-1path1-4summ2-8, pat1-1path2-1summ2-8, path1-4path2-1summ2-8, and pat1-1path1-4path2-1summ2-8 using the NucleoSpin® RNA Plant kit (Machery-Nagel). RNA quality, library preparation and sequencing were performed by BGI. RNA-seq reads were mapped to the Arabidopsis thaliana TAIR10 reference genome with STAR (version 2.5.1b) using 2-pass alignment mode (Dobin et al., 2013). The read counts for each gene were detected using HTSeq (version 0.5.4p3) (Anders et al., 2015). The Araport11 annotation was used for both mapping and read counting. The counts were normalized using the TMM normalization from edgeR package in R (Robinson et al., 2010). PCA analysis were performed using IPython notebook (Wang and Ma’ayan, 2016). Prior to statistical testing the data was voom transformed and then the differential expression between the sample groups was calculated with limma package in R (Law et al., 2014; Ritchie et al., 2015). Genes with fold change ≥2 or ≤-2 and P-value ≤0.05 are listed in Data File S1. Functional Annotation Tool DAVID Bioinformatics Resources 6.8 has been used for GO term analysis for the differently expressed genes using detected genes as background (Huang et al., 2009).

Cloning and transgenic lines

PATs promoter sequences with 5’ HindIII and 3’ XbaI linkers were amplified from Col-0 genomic DNA and cloned in plasmid pGWB515 to make pGWB515-PATsprom (Nakagawa et al., 2007). The Venus sequence without stop codon was amplified from pEN-L1-Venus-L2 (Mylle et al., 2013) and cloned in pGWB515-PATsprom. PAT genes were amplified from Col-0 genomic DNA and cloned into pENTR-D-TOPO (Invitrogen). The entry clones were combined with pGWB515-PATsprom and pGWB515-PATsprom-Venus to obtain N-terminal HA and Venus tags respectively (Nakagawa et al., 2007). pENTR-D-TOPO-GUS (Invitrogen) and pENTR-D-TOPO-LSM1 (Roux et al., 2015) were combined to pK7WGY2.0 (Karimi et al., 2007) to obtain N-terminal YFP tags. These fusions were transformed into Agrobacterium tumefaciens strain GV3101 for transient and stable expression. Transformants were selected on hygromycin (30 mg/L) MS agar, and survivors tested for transcript expression by qRT-PCR and protein expression by immuno-blotting. Primers for cloning are provided in Table S2.

Transient expression, protein extraction and co-IP in Nicotiana benthamiana

A. tumefaciens strains carrying PAT fusions were grown in YEP medium supplemented with appropriate antibiotics overnight. Cultures were centrifuged and re-suspended in buffer (10mM MgCl2, 10 mM MES-K (pH 5.6), 100 μM acetosyringone) to OD600=0.8. A. tumefaciens strains carrying PATs-HA and YFP-LSM1 or YFP-GUS were mixed 1:1 and infiltrated into 3-week-old N. benthamiana leaves. Leaf samples for protein extraction and immunoprecipitation were collected 3 days post infiltration (dpi). Tissues for protein extraction were ground in liquid nitrogen and IP buffer (50mM Tris-HCl pH 7.5; 150 mM NaCl; 5 % (v/v) glycerol; 1 mM EDTA; 0.1%(v/v) NP40; 10 mM DTT; protease inhibitor cocktail (Roche); Phosstop (Roche)) added at 2mL/g tissue powder. Following 20 min centrifugation at 4°C and 13000 rpm, sample supernatants were adjusted to 2mg/ml protein and incubated 4 hours at 4°C with GFPTrap-A beads (Chromotek). Beads were washed 4 times with wash buffer (20 mM Tris pH 7.5; 150m M NaCl; 0.1 % (v/v) NP40 before adding 4×SDS buffer (novex)) and denatured by heating at 95°C for 5 min.

Protein extraction, SDS-PAGE and immunoblotting

Tissue was ground in liquid nitrogen and 4×SDS buffer (novex) was added and heated at 95°C for 5 min, cooled to room temperature for 10min, samples were centrifuged 5min at 13000 rpm. Supernatants were separated on 10% SDS-PAGE gels, electroblotted to PVDF membrane (GE Healthcare), blocked in 5% (w/v) milk in TBS-Tween 20 (0.1%, v/v) and incubated 1hr to overnight with primary antibodies (anti-GFP (AMS Biotechnology 1:5.000, anti-HA 1:1,000 (Santa Cruz)). Membranes were washed 3 × 10 min in TBS-T (0.1%) before 1hr incubation in secondary antibodies (anti-rabbit or anti-mouse HRP or AP conjugate (Promega; 1: 5.000)). Chemiluminescent substrate (ECL Plus, Pierce) was applied before camera detection. For AP-conjugated primary antibodies, membranes were incubated in NBT/BCIP (Roche) until bands were visible.

Figure

Fig S1.
Fig S1. Characterization of pat mutants generated using CRISPR/CAS9 system.

(A), 6 week-old plants of summ2-8, pat1-1summ2-8, path1-5path2-2summ2-8 and pat1-1path1-5path2-2summ2-8 grown in soil in chamber with 8/16hr light/dark at 21°C. One representative plant for each line is shown. The scale bar indicates 1cm. (B) sequence of independent path1 and path2 mutations. Western blots detecting the expression of PATH1 and PATH2 (C) and PAT1 (E) fusions with N-Venus and growth phenotype (D&F) of complemented lines. One representative plant for each line is shown. The scale bar indicates 1cm.

Fig S2.
Fig S2. Analysis of differently expressed genes in pat triple mutants.

Gene ontology analysis of transcripts upregulated (A) or downregulated (B) in 6-week-old plants of mRNA decay deficient mutant pat1-1path1-4path2-1summ2-8 compared to summ2-8.

View this table:
Table S1. Mutants used in this study
View this table:
Table S2. Primers used in this study

Acknowledgments

We thank Qi-Jun Chen for Phee401. Special thanks to John Mundy for advice throughout the project and critically reading the manuscript. We acknowledge the Bioinformatics and Scientific Computing Facility (VBCF) and Xiaoye Tong for the next-generation sequencing data analysis. This work was supported by the Novo Nordisk Fonden and the Hartmanns Fond to MP (NNF18OC0052967 and A32638) and a PhD scholarship from China Scholarship Council to ZZ (201504910714).

ZZ, MER, and MP conceived and designed the experiments. ZZ, MER, YD and ER performed experiments. ZZ and MP analyzed the data. ZZ and MP wrote the manuscript.

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to MP.

References

  1. ↵
    Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169.
    OpenUrlCrossRefPubMedWeb of Science
  2. ↵
    Balagopal, V., and Parker, R. (2009). Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21, 403–408.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    Bilsborough G.D., Runions A., Barkoulas M., Jenkins H.W., Hasson A., Galinha C., Laufs P., Hay A., Prusinkiewicz P., Tsiantis M. (2011) Model for the regulation of Arabidopsis thaliana leaf margin development. Proc. Natl. Acad. Sci. USA. 108:3424–3429.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Brengues, M., Teixeira, D., and Parker, R. (2005). Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Charenton, C., Gaudon-Plesse, C., Fourati, Z., Taverniti, V., Back, R., Kolesnikova, O., Seraphin, B., and Graille, M. (2017). A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5 ‘-3 ‘mRNA exonuclease in yeast. P Natl Acad Sci USA 114, E9493–E9501.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Chowdhury, A., Kalurupalle, S., and Tharun, S. (2014). Pat1 contributes to the RNA binding activity of the Lsm1-7-Pat1 complex. Rna-a Publication of the Rna Society 20, 1465–1475.
    OpenUrlAbstract/FREE Full Text
  7. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735–743.
    OpenUrlCrossRefPubMedWeb of Science
  8. Coller, J., and Parker, R. (2005). General translational repression by activators of mRNA decapping. Cell 122, 875–886.
    OpenUrlCrossRefPubMedWeb of Science
  9. ↵
    Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T.R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21.
    OpenUrlCrossRefPubMedWeb of Science
  10. ↵
    Duarte, J.M., Cui, L., Wall, P.K., Zhang, Q., Zhang, X., Leebens-Mack, J., Ma, H., Altman, N., and dePamphilis, C.W. (2006). Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol Biol Evol 23, 469–478.
    OpenUrlCrossRefPubMedWeb of Science
  11. ↵
    Essig, K., Kronbeck, N., Guimaraes, J.C., Lohs, C., Schlundt, A., Hoffmann, A., Behrens, G., Brenner, S., Kowalska, J., Lopez-Rodriguez, C., Jemielity, J., Holtmann, H., Reiche, K., Hackermuller, J., Sattler, M., Zavolan, M., and Heissmeyer, V. (2018). Roquin targets mRNAs in a 3 ‘-UTR-specific manner by different modes of regulation. Nat Commun 9.
  12. Franks, T.M., and Lykke-Andersen, J. (2008). The Control of mRNA Decapping and P-Body Formation. Mol Cell 32, 605–615.
    OpenUrlCrossRefPubMedWeb of Science
  13. Garneau, N.L., Wilusz, J., and Wilusz, C.J. (2007). The highways and byways of mRNA decay. Nat Rev Mol Cell Bio 8, 113–126.
    OpenUrlCrossRefPubMed
  14. ↵
    Gatica D, Hu G, Liu X, Zhang N, Williamson PR, Klionsky DJ. (2019) The Pat1-Lsm Complex Stabilizes ATG mRNA during Nitrogen Starvation-Induced Autophagy. Mol Cell. 73(2):314-324.e4.
    OpenUrlCrossRef
  15. ↵
    Huang, D.W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44–57.
    OpenUrlCrossRefPubMedWeb of Science
  16. Jang, G.J., Yang, J.Y., Hsieh, H.L., and Wu, S.H. (2019). Processing bodies control the selective translation for optimal development of Arabidopsis young seedlings. P Natl Acad Sci USA 116, 6451–6456.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Karimi, M., Depicker, A., and Hilson, P. (2007). Recombinational cloning with plant gateway vectors. Plant Physiol 145, 1144–1154.
    OpenUrlFREE Full Text
  18. ↵
    Law, C.W., Chen, Y.S., Shi, W., and Smyth, G.K. (2014). voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15.
  19. ↵
    Léo Serra, Catherine Perrot-Rechenmann.(2020) Spatiotemporal control of cell growth by CUC3 shapes leaf margins. Development 15, 147 (6): dev183277.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Lobel, J.H., Tibble, R.W., Gross, J.D. (2019). Pat1 activates late steps in mRNA decay by multiple mechanisms. P Natl Acad Sci USA 116 (47), 23512–23517
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Merret, R., Descombin, J., Juan, Y.T., Favory, J.J., Carpentier, M.C., Chaparro, C., Charng, Y.Y., Deragon, J.M., and Bousquet-Antonelli, C. (2013). XRN4 and LARP1 Are Required for a Heat-Triggered mRNA Decay Pathway Involved in Plant Acclimation and Survival during Thermal Stress. Cell Rep 5, 1279–1293.
    OpenUrlCrossRefPubMedWeb of Science
  22. ↵
    Miyamoto, T., Furusawa, C., and Kaneko, K. (2015). Pluripotency, Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation. Plos Comput Biol 11.
  23. ↵
    Mylle, E., Codreanu, M.C., Boruc, J., and Russinova, E. (2013). Emission spectra profiling of fluorescent proteins in living plant cells. Plant Methods 9.
  24. ↵
    Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104, 34–41.
    OpenUrlCrossRefPubMedWeb of Science
  25. ↵
    Newman, R., Ahlfors, H., Saveliev, A., Galloway, A., Hodson, D.J., Williams, R., Besra, G.S., Cook, C.N., Cunningham, A.F., Bell, S.E., and Turner, M. (2017). Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1. Nat Immunol 18, 683-+.
    OpenUrlCrossRef
  26. ↵
    Ozgur, S., Chekulaeva, M., and Stoecklin, G. (2010). Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies. Mol Cell Biol 30, 4308–4323.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Perea-Resa, C., Hernandez-Verdeja, T., Lopez-Cobollo, R., Castellano, M.D., and Salinas, J. (2012). LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development. Plant Cell 24, 4930–4947.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Perea-Resa, C., Carrasco-Lopez, C., Catala, R., Tureckova, V., Novak, O., Zhang, W.P., Sieburth, L., Jimenez-Gomez, J.M., and Salinas, J. (2016). The LSM1-7 Complex Differentially Regulates Arabidopsis Tolerance to Abiotic Stress Conditions by Promoting Selective mRNA Decapping. Plant Cell 28, 505–520.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen, B., Nielsen, H.B., Lacy, M., Austin, M.J., Parker, J.E., Sharma, S.B., Klessig, D.F., Martienssen, R., Mattsson, O., Jensen, A.B., and Mundy, J. (2000). Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103, 1111–1120.
    OpenUrlCrossRefPubMedWeb of Science
  30. ↵
    Sasidharan, R., Chinnappa, C.C., Staal, M., Elzenga, J.T.M., Yokoyama, R., Nishitani, K., Voesenek, L.A.C.J., Pierik, R. (2020) Light Quality-Mediated Petiole Elongation in Arabidopsis during Shade Avoidance Involves Cell Wall Modification by Xyloglucan Endotransglucosylase/Hydrolases, Plant Phys, 154(2): 978–990
    OpenUrl
  31. ↵
    Ritchie, M.E., Phipson, B., Wu, D., Hu, Y.F., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43.
  32. ↵
    Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140.
    OpenUrlCrossRefPubMedWeb of Science
  33. ↵
    Roux, M.E., Rasmussen, M.W., Palma, K., Lolle, S., Regue, A.M., Bethke, G., Glazebrook, J., Zhang, W.P., Sieburth, L., Larsen, M.R., Mundy, J., and Petersen, M. (2015). The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2. Embo J 34, 593–608.
    OpenUrlAbstract/FREE Full Text
  34. Sorenson, R.S., Deshotel, M.J., Johnson, K., Adler, F.R., and Sieburth, L.E. (2018). Arabidopsis mRNA decay landscape arises from specialized RNA decay substrates, decapping - mediated feedback, and redundancy. P Natl Acad Sci USA 115, E1485–E1494.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Tharun S. Lsm1-7-Pat1 complex: a link between 3’ and 5’-ends in mRNA decay? (2009) RNA Biol. 6(3):228–32.
    OpenUrlCrossRefPubMedWeb of Science
  36. ↵
    van Wijk R., Zhang Q., Zarza X., Lamers M., Marquez F. R., Guardia A., Scuffi D., García-Mata C., Ligterink W., Haring M. A., Laxalt A. M., and Munnik T. (2018). Role for Arabidopsis PLC7 in Stomatal Movement, Seed Mucilage Attachment, and Leaf Serration. Front Plant Sci. 9, 1721
    OpenUrl
  37. ↵
    Wang, Z.P., Xing, H.L., Dong, L., Zhang, H.Y., Han, C.Y., Wang, X.C., and Chen, Q.J. (2015). Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol 16.
  38. ↵
    Wang Z and Ma’ayan A. (2016) An open RNA-Seq data analysis pipeline tutorial with an example of reprocessing data from a recent Zika virus study. F1000Research, 5:1574
  39. ↵
    Xie, Y., Liu, Y., Wang, H. et al. (2017) Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis. Nat Commun 8, 348.
    OpenUrlCrossRefPubMed
  40. ↵
    Xiong, Y. & Jiao, Y. The Diverse Roles of Auxin in Regulating Leaf Development. (2019) Plants 8, 243.
    OpenUrl
  41. ↵
    Xu, J., and Chua, N.H. (2009). Arabidopsis Decapping 5 Is Required for mRNA Decapping, P-Body Formation, and Translational Repression during Postembryonic Development. Plant Cell 21, 3270–3279.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Xu, J., and Chua, N.H. (2012). Dehydration stress activates Arabidopsis MPK6 to signal DCP1 phosphorylation. Embo J 31, 1975–1984.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Xu, J., Yang, J.Y., Niu, Q.W., and Chua, N.H. (2006). Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development. Plant Cell 18, 3386–3398.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Zhang P, Berardini TZ, Ebert D, Li Q, Mi H, Muruganujan A, Prithvi T, Reiser L, Sawant S, Thomas PD, Huala E. (2020). PhyloGenes: An online phylogenetics and functional genomics resource for plant gene function inference. Plant Direct. 4(12):e00293
    OpenUrl
  45. ↵
    Zhang, Z.B., Wu, Y.L., Gao, M.H., Zhang, J., Kong, Q., Liu, Y.A., Ba, H.P., Zhou, J.M., and Zhang, Y.L. (2012). Disruption of PAMP-Induced MAP Kinase Cascade by a Pseudomonas syringae Effector Activates Plant Immunity Mediated by the NB-LRR Protein SUMM2. Cell Host Microbe 11, 253–263.
    OpenUrlCrossRefPubMedWeb of Science
  46. ↵
    Zuo, Z., Roux, M.E., Sæmundsson, H.P., Müller, M., Munne Bosch, S., and Petersen, M. (2021). The Arabidopsis thaliana mRNA decay factor PAT1 functions in osmotic stress responses and decaps ABA-responsive genes. Febs Lett, 595:253–263.
    OpenUrl
  47. ↵
    Zuo, Z., Roux, M.E., Rodriguez, E., and Petersen, M. (2022a). mRNA decapping factors LSM1 and PAT paralogs are invol ved in TuMV viral infection. Mol Plant Microbe Interact. 2022 Feb; 35(2): 125–130.
    OpenUrl
Back to top
Posted July 06, 2022.
Download PDF

Supplementary Material

Email
PAT mRNA decapping factors function specifically and redundantly during development in Arabidopsis
Zhangli Zuo, Milena Edna Roux, Yasin F. Dagdas, Eleazar Rodriguez, Morten Petersen
bioRxiv 2022.07.06.498930; doi: https://doi.org/10.1101/2022.07.06.498930
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area