HEM1 Is A Translational Regulator Of Plant Immunity

Plant diseases have always been a significant problem for agriculture,posing a severe threat to food security.In recent years,climate extremes have increased the frequency of plant diseases.Therefore,it is important to study the molecular mechanisms of plant immunity to help agriculture breed better disease-resistant crops without fitness costs.Transcriptional regulation is necessary but not always sufficient for plant immunity.Recent studies have shown that translational reprogramming is a fundamental layer of immune regulation.The TBF1 5’leader contains upstream open reading frames（uORFs）to be immune responsive.This thesis generated a transgenic Arabidopsis thaliana line with the uORFs _TBF1-LUC translational reporter using the TBF1 5’leader to control the translation of firefly luciferase（Luciferase,LUC）.A forward genetic screen was performed,and the translation regulator HEM1 was successfully identified.The results demonstrate that HEM1,as a translation regulator,promotes cell survival and tissue health by inhibiting ETI-associated cell death caused by the immune gene over-translation.The main findings of the study are as follows,（1）The dst5 mutant was found in a forward genetic screen with enhanced LUC activity under normal growth conditions.The dst5 mutant was analyzed by whole-genome resequencing,genetic complementation verification,and measuring LUC translation efficiency.These steps confirmed that the HEM1 was the causal gene for the dst5mutant phenotype.After ETI activation,hem1 exhibited a more rapid ETI-associated cell death than WT,suggesting that HEM1 is a negative ETI cell death regulator.Ribo-seq sequencing data revealed that genes positively dependent on HEM1 were involved in determining m RNA fate,which helped maintain translation homeostasis.In contrast,genes negatively dependent on HEM1 were mainly enriched in hypoxia and defense responses after ETI treatment.During ETI activation,Some immune-related genes with low translation efficiency in WT were significantly increased in the hem1 mutant.Futher studies suggests that the free translation of those immune genes leads to accelerated cell death in hem1 during ETI activation.It demonstrates that the loss of HEM1 provokes a global translational perturbation and alleviates the translational restriction of pro-death immune genes.（2）We used immunoprecipitation and mass spectrometry and identified 257 HEM1-interacting proteins.HEM1 interacts with many translation factors,such as subunits of e IF4F,e IF2,e IF3,and e IF5B,and some release factors,in addition to actin nucleation-related genes.Analyzing HEM1’s protein sequence found that the majority consisted of the classical Nckap1 domain,whose sequence and structural similarities were shared across higher eukaryotes.An evolutionary tree of HEM1 homologs revealed that HEM proteins were present as two discrete subfamily members（HEM1/2）in animals,while plant species had only one member and varied through gene copy numbers in polyploids.Compared with the animal HEM1/2 proteins,all plant-derived HEM1 proteins had a unique carboxy-terminal extension predicted to be a low-complexity domain（LCD）.Split-luciferase complementation assay（SLCA）examined the interactions among HEM1,HEM1ΔLCD and LCD with successfully cloned 77 translation factors.SLCA detected 35 strongly pairwise interactions.These interactions were dependent on LCD.（3）HEM1 is a phase separation protein that forms condensates dependent on the plant-specific LCD in vitro and in vivo.This LCD mediates the formation of HEM1condensates with translation factors in response to immune signals.Using Bi FC and co-localization analysis,We found that HEM1 interacted with translation factors in HEM1 condensates.It further suggests that HEM1 regulates translational reprogramming through phase separation by forming condensates.The minimal size of HEM1 pertaining to the role of actin was mapped through the consecutive deletion of the LCD-containing region using a CRISPR-walking method.Only removing the exact LCD region（ΔLCD）increased LUC activity in the translational reporter without the intervention of actin nucleation,as indicated by normal trichome morphology.However,it showed the same translational regulation and immune response phenotypes as the complete deletion mutant hem1,indicating that HEM1 utilizes a plant-specific LCD to mediate translational regulation and ETI-induced cell death.In summary,HEM1 forms condensates by interacting with many translation factors through the plant-specific LCD domain,which plays an essential role in plant immunity.The formation of HEM1 condensates effectively inhibits the excessive translation of immune genes,thus providing a trade-off between tissue health and disease resistance.This mechanism constitutes a feedback mechanism to inhibit immune over-activation,thus preventing tissue damage caused by excessive immune activation in plants.The mechanism provides mechanistic support for engineering disease-resistant,high-yield,high-quality crops.