The PELOTA-HBS1 Complex Orchestrates mRNA Translation Surveillance and PDK1-mediated Plant Growth and Development

The quality control system for messenger RNA is fundamental for cellular activities in eukaryotes. To elucidate the molecular mechanism of 3’-Phosphoinositide-Dependent Protein Kinase1 (PDK1), an essential regulator throughout growth and development of eukaryotes, a forward genetic approach was employed to screen for suppressors of the loss-of-function T-DNA insertional pdk1.1 pdk1.2 double mutant in Arabidopsis. Notably, the severe growth attenuation of pdk1.1 pdk1.2 is rescued by sop21 (suppressor of pdk1.1 pdk1.2) that harbours a loss-of-function mutation in PELOTA1 (PEL1). PEL1 is a homologue of mammalian PELOTA and yeast DOM34, which form a heterodimeric complex with the GTPase HBS1, responsible for ribosome rescue to assure the quality and fidelity of mRNA molecules. Genetic analysis further reveals that the dysfunction of PEL1-HBS complex fails to degrade the T-DNA-disrupted, truncated but functional PDK1 transcripts, thus rescuing pdk1.1 pdk1.2. Our studies demonstrate the functionality and identify the essential functions of a homologous PELOTA-HBS1 complex in higher plant, and provide novel insights into the mRNA quality control mechanism.

3 Introduction hbs1 plants. The above observations led us to test whether the increased levels of truncated 217 PDK1.1 or PDK1.2 could explain for the rescue of pdk1.1 pdk1.2. 218 By transforming a mCherry-fused PDK1. 1N (1-480 aa) (12) driven by pPDK1.1 219 promoter into pdk1.1 pdk1.2, a partial rescue was observed (Fig. 2H). In addition,   9). In addition, PEL1-YFP exhibits nuclear distribution as well, which was undetectable 238 for PDK1s and HBS1 ( Fig. 2A; Supplementary Fig. 9A, B). We speculate that these proteins 239 might present differential functions beyond the potential common pathways. 240 A tandem mass tag (TMT)-based comparative proteomics analysis was then performed 241 using shoots and roots of two-week-old seedlings, and 6995 and 8137 proteins were 242 quantified in shoots and roots respectively. We studied shoots and roots separately, because proteins and were speculated being responsible for the defective growth of pdk1.1 pdk1.2. 248 Most RCE proteins showed increased levels (84 of 102 proteins) in pdk1.1 pdk1.2, 9 suggesting that PDK1 deficiency led to the enhanced recycling of ribosomes and hence the 250 increased abundance of RCE proteins, further confirming that PDK1-mediated regulation of 251 PELOTA-HBS1 complex is crucial to maintain the normal recycling of ribosomes and 252 protein synthesis. 253 KEGG analysis of RCE proteins revealed the enriched metabolic pathways including 254 lipids, carbohydrates, phenylpropanoid and amino acids, and involvement in multiple 255 developmental processes and environmental adaptation (Table 1) (Table 1). Meanwhile, some 264 abiotic stress-related proteins, especially cold acclimation/responsive proteins (51-54) 265 significantly increased in pdk1.1 pdk1.2 shoots. This is consistent with the previous studies 266 showing that PDK1 positively regulates basal resistance in rice (10) and PDK1 is required 267 for P. indica-induced growth promotion (11), and the rice PELOTA protein is involved in 268 bacterial leaf blight resistance (55).

269
Notably, the TMT-based comparative proteomics analysis also showed that PEL1 and 270 HBS1 presented unchanged protein abundance in pdk1.1 pdk1.2 ( Supplementary Fig. 10A Fig. 11A, B). Therefore, it is very unlikely that changes of these AGC 276 substrates might account for the rescue of pdk1.1 pdk1.2 by the pel1 mutation.
10 Discussion 278 279 PDK1 is highly conserved in eukaryotes and is essential for growth and development of 280 various organisms. PDK1 deficiency results in severe growth defects or even lethality, which 281 have impeded the studies on the underlying molecular mechanism, especially in mammals.

282
Taking advantage of plant genetics and by screening for the suppressors that rescue the 283 growth defects of T-DNA insertional pdk1.1 pdk1.2 mutants, we here identify PEL1, which 284 is a component of the PEL1-HBS1 mRNA surveillance complex and is essential for the 285 mRNA quality control during protein translation. The mechanism for the pel1 and hbs1 286 mutations suppressing the pdk1.1 pdk1.2 phenotype is their inability to degrade the aberrant 287 mRNAs, leading to the production of truncated but functional PDK1 proteins (Fig. 5A, B). 288 Our studies reveal that the PEL1-HBS1 complex coordinates the ribosome rescue and 289 protein biosynthesis (Fig. 6A Storage proteins are actively synthesised at rough ER as precursor forms and then are 331 transported into protein storage vacuole (PSV) during seed maturation (68). In higher plants, 332 seed storage proteins are deposited in PSVs of dry seeds as a source of nitrogen for growth 333 after seed germination (68,69). Accumulation of seed storage proteins and nutrient 334 reserve-related proteins in pdk1.1 pdk1.2 shoots may result in the altered vegetative growth.

335
This indicates that PDK1 represses seed storage proteins and nutrient reserve-related 336 proteins in the vegetative tissues or the nitrogen utilization after seed germination. 337 It is noteworthy that the pdk1.1 pdk1.2 phenotype is not fully rescued by pel1 mutations, 338 or by a truncated PDK1.1N transgene. We speculate that the truncated PDK1 protein only
Length of primary root and number of emerged lateral roots of 2-week-old seedlings were calculated. Data are presented as means ± SD (n > 30). n = 56, 65, 52, 62, and 30, respectively. Different letters represent significant difference, P < 0.05, by one-way analysis of variance (ANOVA) with a Tukey multiple comparison test. E.
Number of emerged lateral roots of two-week-old seedlings were counted. and statistically analyzed by student's t-test (**, p < 0.01). Data are presented as means ± SD (n > 30). n = 61, 65, 63, 63, and 52, respectively. Different letters represent significant difference, P < 0.01, by one-way ANOVA with a Tukey multiple comparison test.   Kong et al., Figure 5 A B

Arabidopsis.
Heat map displayed the abundance of 54 RCEs (restored CE proteins) in shoots (A) and 49 RCEs in roots (B) of wild type Col-