TOR coordinates cytokinin and gibberellin signals mediating development and defense.

Plants constantly perceive and process environmental signals and balance between the energetic demands of growth and defense. Growth arrest upon pathogen attack was previously suggested to result from a redirection of the plants' metabolic resources towards the activation of plant defense. The energy sensor Target of Rapamycin (TOR) kinase is a conserved master coordinator of growth and development in all eukaryotes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms by which TOR may potentially regulate the relationship between these two modalities. The plant hormones cytokinin (CK) and gibberellin (GA) execute various aspects of plant development and defense. The ratio between CK and GA was reported to determine the outcome of developmental programmes. Here, investigating the interplay between TOR-mediated development and TOR-mediated defense in tomato, we found that TOR silencing resulted in rescue of several different aberrant developmental phenotypes, demonstrating that TOR is required for the execution of developmental cues. In parallel, TOR inhibition enhanced immunity in genotypes with a low CK/GA ratio but not in genotypes with a high CK/GA ratio. TOR-inhibition mediated disease resistance was found to depend on developmental status, and was abolished in strongly morphogenetic leaves, while being strongest in mature, differentiated leaves. CK repressed TOR activity, suggesting that CK-mediated immunity may rely on TOR downregulation. At the same time, TOR activity was promoted by GA, and TOR silencing reduced GA sensitivity, indicating that GA signalling requires normal TOR activity. Our results demonstrate that TOR likely acts in concert with CK and GA signalling, executing signalling cues in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could regulate the required outcome of development-defense prioritisation.

SlRLK-like is a malectin-like domain protein affecting localization and abundance of LeEIX2 receptor resulting in suppression of EIX-induced immune responses.

The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe-associated molecular patterns. Plant PRRs are either receptor-like kinases (RLKs), which have an extracellular domain for ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor-like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene-inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two-hybrid screen and found a tomato protein that we termed SlRLK-like. The interaction of LeEIX2 with SlRLK-like was verified using co-immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK-like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK-like using the CRISPR/Cas9 system increased EIX-induced ethylene production and 1-aminocyclopropane-1-carboxylate synthase (SlACS2) gene expression in tomato. Co-expression of SlRLK-like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum-associated degradation process. Notably, truncation of SlRLK-like protein revealed that the malectin-like domain is sufficient and essential for its function. Moreover, SlRLK-like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)-induced burst of reactive oxygen species. In addition, SlRLK-like co-expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK-like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses.

Organelle-Targeted BODIPY Photocages: Visible-Light-Mediated Subcellular Photorelease.

Photocaging facilitates non-invasive and precise spatio-temporal control over the release of biologically relevant small- and macro-molecules using light. However, sub-cellular organelles are dispersed in cells in a manner that renders selective light-irradiation of a complete organelle impractical. Organelle-specific photocages could provide a powerful method for releasing bioactive molecules in sub-cellular locations. Herein, we report a general post-synthetic method for the chemical functionalization and further conjugation of meso-methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)-, lysosome-, and mitochondria-targeted derivatives. We also demonstrate that 2,4-dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.

The intracellular nucleotide-binding leucine-rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception.

Plant recognition and defence against pathogens employs a two-tiered perception system. Surface-localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR required for hypersensitive response-associated cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.

Sterol-dependent induction of plant defense responses by a microbe-associated molecular pattern from Trichoderma viride.

Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.

The function of EHD2 in endocytosis and defense signaling is affected by SUMO.

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.

Abolishing ARF8A activity promotes disease resistance in tomato.

Auxin response factors (ARFs) are a family of transcription factors that regulate auxin-dependent developmental processes. Class A ARFs function as activators of auxin-responsive gene expression in the presence of auxin, while acting as transcriptional repressors in its absence. Despite extensive research on the functions of ARF transcription factors in plant growth and development, the extent, and mechanisms of their involvement in plant resistance, remain unknown. We have previously reported that mutations in the tomato AUXIN RESPONSE FACTOR8 (ARF8) genes SlARF8A and SlARF8B result in the decoupling of fruit development from pollination and fertilization, leading to partial or full parthenocarpy and increased yield under extreme temperatures. Here, we report that fine-tuning of SlARF8 activity results in increased resistance to fungal and bacterial pathogens. This resistance is mostly preserved under fluctuating temperatures. Thus, fine-tuning SlARF8 activity may be a potent strategy for increasing overall growth and yield.

Sensing of gene expression in live cells using electrical impedance spectroscopy and DNA-functionalized gold nanoparticles.

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the ß-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities.

In Vivo Plant Bio-Electrochemical Sensor Using Redox Cycling.

This work presents an in vivo stem-mounted sensor for Nicotiana tabacum plants and an in situ cell suspension sensor for Solanum lycopersicum cells. Stem-mounted sensors are mechanically stable and less sensitive to plant and air movements than the previously demonstrated leaf-mounted sensors. Interdigitated-electrode-arrays with a dual working electrode configuration were used with an auxiliary electrode and an Ag/AgCl quasi-reference electrode. Signal amplification by redox cycling is demonstrated for a plant-based sensor responding to enzyme expression induced by different cues in the plants. Functional biosensing is demonstrated, first for constitutive enzyme expression and later, for heat-shock-induced enzyme expression in plants. In the cell suspension with redox cycling, positive detection of the enzyme ß-glucuronidase (GUS) was observed within a few minutes after applying the substrate (pNPG, 4-Nitrophenyl ß-D-glucopyranoside), following redox reactions of the product (p-nitrophenol (pNP)). It is assumed that the initial reaction is the irreversible reduction of pNP to p-hydroxylaminophenol. Next, it can be either oxidized to p-nitrosophenol or dehydrated and oxidized to aminophenol. Both last reactions are reversible and can be used for redox cycling. The dual-electrode redox-cycling electrochemical signal was an order of magnitude larger than that of conventional single-working electrode transducers. A simple model for the gain is presented, predicting that an even larger gain is possible for sub-micron electrodes. In summary, this work demonstrates, for the first time, a redox cycling-based in vivo plant sensor, where diffusion-based amplification occurs inside a tobacco plant's tissue. The technique can be applied to other plants as well as to medical and environmental monitoring systems.

Members of the tomato NRC4 h-NLR family augment each other in promoting basal immunity.

Plants possess an efficient, two-tiered immune system to combat pathogens and pests. Several decades of research have characterized different features of these two well-known tiers, PTI and ETI (Pattern/ Effector-triggered Immunity). NLR (Nucleotide-binding domain Leucine-rich Repeat) receptors have been found to link PTI to ETI, and be required for full potentiation of plant immune responses in several systems. Intra-cellular helper-NLRs (h-NLRs) mediate ETI and have been focused on extensively in recent research. Previously, we investigated the roles of the h-NLR SlNRC4a in tomato immunity, finding that a specific mutation in this gene results in gain of function constitutive defense activation and broad disease resistance. Deletion of the entire NRC4 clade, which contains 3 genes, can compromise tomato immunity. Here, we decided to investigate the role of an additional clade member, SlNRC4b, in basal immunity. We generated a gain of function mutant in SlNRC4b using CRISPR-Cas9, as well as a double gain of function mutant in both genes. Similarly to the slnrc4a mutant, a slnrc4b mutant also possessed increased basal immunity and broad spectrum disease resistance. The double mutant displayed additive effects in some cases, with significant increases in resistance to fungal phytopathogens as compared with each of the single mutants. Our work confirms that the NRC4 family h-NLRs are important in the plant immune system, suggesting that this gene family has the potential to be promising in targeted agricultural adaptation in the Solanaceae family, promoting disease resistance and prevention of yield loss to pathogens.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2.

Extracellular leucine-rich repeat (LRR) receptor-like proteins (RLPs) represent a unique class of cell-surface receptors, as they lack a functional cytoplasmic domain. Our knowledge of how RLPs that do not contain a kinase or Toll domain function is very limited. The tomato RLP receptor LeEix2 signals to induce defense responses mediated by the fungal protein ethylene-inducing xylanase (EIX). The movement of FYVE-positive endosomes before and after EIX application was examined using spinning disc confocal microscopy. We found that while FYVE-positive endosomes generally observe a random movement pattern, following EIX application a subpopulation of FYVE-positive endosomes follow a directional movement pattern. Further, cellular endosomes travel greater distances at higher speeds following EIX application. Time-course experiments conducted with specific inhibitors demonstrate the involvement of endosomal signaling in EIX-triggered defense responses. Abolishing the existence of endosomes or the endocytic event prevented EIX-induced signaling. Endocytosis/endosome inhibitors, such as Dynasore or 1-butanol, inhibit EIX-induced signaling. Moreover, treatment with Endosidin1, which inhibits an early step in plasma membrane/endosome trafficking, enhances the induction of defense responses by EIX. Our data indicate a distinct endosomal signaling mechanism for induction of defense responses in this RLP system.

TOR inhibition primes immunity and pathogen resistance in tomato in a salicylic acid-dependent manner.

All organisms need to sense and process information about the availability of nutrients, energy status, and environmental cues to determine the best time for growth and development. The conserved target of rapamycin (TOR) protein kinase has a central role in sensing and perceiving nutritional information. TOR connects environmental information about nutrient availability to developmental and metabolic processes to maintain cellular homeostasis. Under favourable energy conditions, TOR is activated and promotes anabolic processes such as cell division, while suppressing catabolic processes. Conversely, when nutrients are limited or environmental stresses are present, TOR is inactivated, and catabolic processes are promoted. Given the central role of TOR in regulating metabolism, several previous works have examined whether TOR is wired to plant defence. To date, the mechanisms by which TOR influences plant defence are not entirely clear. Here, we addressed this question by testing the effect of inhibiting TOR on immunity and pathogen resistance in tomato. Examining which hormonal defence pathways are influenced by TOR, we show that tomato immune responses and disease resistance to several pathogens increase on TOR inhibition, and that TOR inhibition-mediated resistance probably requires a functional salicylic acid, but not jasmonic acid, pathway. Our results support the notion that TOR is a master regulator of the development-defence switch in plants.

Removal of carbamazepine, venlafaxine and iohexol from wastewater effluent using coupled microalgal-bacterial biofilm.

We evaluated the removal capacity of a coupled microalgal-bacterial biofilm (CMBB) to eliminate three recalcitrant pharmaceuticals. The CMBB's efficiency, operating at different biofilm concentrations, with or without light, was compared and analyzed to correlate these parameters to pharmaceutical removal and their effect on the microorganism community. Removal rates changed with changing pharmaceutical and biofilm concentrations: higher biofilm concentrations presented higher removal. Removal of 82-94% venlafaxine and 18-51% carbamazepine was obtained with 5 days of CMBB treatment. No iohexol removal was observed. Light, microorganism composition, and dissolved oxygen concentration are essential parameters governing the removal of pharmaceuticals and ammonia. Chlorophyll concentration increased with time, even in the dark. Three bacterial phyla were dominant: Proteobacteria, Bacteroidetes and Firmicutes. The dominant eukaryotic supergroups were Archaeplastida, Excavata and SAR. A study of the microorganisms' community indicated that not only do the species in the biofilm play an important role; environment, concentration and interactions among them are also important. CMBB has the potential to provide low-cost and sustainable treatment for wastewater and recalcitrant pharmaceutical removal. The microenvironments on the biofilm created by the microalgae and bacteria improved treatment efficiency.

Dynamin-Related Proteins Enhance Tomato Immunity by Mediating Pattern Recognition Receptor Trafficking.

Pattern recognition receptor (PRR) trafficking to the plasma membrane and endocytosis plays a crucial role in pattern triggered immunity (PTI). Dynamin-related proteins (DRPs) participate in endocytosis and recycling. In Arabidopsis, DRP1 and DRP2 are involved in plasma membrane scission during endocytosis. They are required for the PRR FLS2 endocytosis induction and PTI activation after elicitation with flg22, the MAMP recognized by FLS2. In tomato, SlDRP2A regulates the PRR LeEIX2 endocytosis and PTI activation in response to EIX, the MAMP recognized by LeEIX2. However, it is unknown if other DRPs participate in these processes. Taking advantage of bioinformatics tools, we selected SlDRP2B among the eight DRP2 tomato orthologues to study its functionality in trafficking and plant immunity. Through transient expression of SlDRP1B and its dominant-negative mutant on Nicotiana benthamiana and Nicotiana tabacum, we analyzed SlDRP1B function. We observed that SlDRP1B is physically associated with the LeEIX2 and modifies LeEIX2 trafficking, increasing its presence in endosomes. An enhancement of EIX-elicitated defense responses accompanies the role of SlDRP1B on LeEIX endocytosis. In addition, SlDRP1B overexpression enhanced flg22-elicited defense response. With these results, we conclude that SlDRP1B regulates PRR trafficking and, therefore, plant immunity, similarly to the SlDRP2A role.

BAK1 is required for the attenuation of ethylene-inducing xylanase (Eix)-induced defense responses by the decoy receptor LeEix1.

Elicitor recognition plays a key role in the reaction of plants to pathogens and the induction of plant defense responses. Furthermore, plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. Ethylene-inducing xylanase (Eix) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). The Eix receptors (LeEix1 and LeEix2) belong to a superclade of leucine-rich repeat receptor-like proteins (RLP) with a signal for receptor-mediated endocytosis, which was shown to be essential for proper induction of defense responses. Both receptors are able to bind Eix, while only LeEix2 mediates defense responses. Here we demonstrate that LeEix1 heterodimerizes with LeEix2 upon application of the Eix elicitor. We show that LeEix1 attenuates Eix-induced internalization and signaling of the LeEix2 receptor. Furthermore, we demonstrate, using yeast two-hybrid and in planta bimolecular fluorescence complementation assays, that the brassinosteroid co-receptor, BAK1, binds LeEix1 but not LeEix2. In BAK1-silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires BAK1.

Towards optimization of plant cell detection in suspensions using impedance-based analyses and the unified equivalent circuit model.

An improved approach for comparative study of plant cells for long term and continuous monitoring using electrical impedance spectroscopy is demonstrated for tomato and tobacco plant cells (MSK8 and BY2) in suspensions. This approach is based on the locations and magnitudes of defining features in the impedance spectra of the recently reported unified equivalent circuit model. The ultra-wide range (4 Hz to 20 GHz) impedance spectra of the cell lines were measured using custom probes, and were analyzed using the unified equivalent circuit model, highlighting significant negative phase peaks in the ~ 1 kHz to ~ 10 MHz range. These peaks differ between the tomato and tobacco cells, and since they can be easily defined, they can potentially be used as the signal for differentiating between different cell cultures or monitoring them over time. These findings were further analysed, showing that ratios relating the resistances of the media and the resistance of the cells define the sensitivity of the method, thus affecting its selectivity. It was further shown that cell agglomeration is also an important factor in the impedance modeling in addition to the overall cell concentration. These results can be used for optimizing and calibrating electrical impedance spectroscopy-based sensors for long term monitoring of cell lines in suspension for a given specific cell and media types.

Electrical impedance spectroscopy of plant cells in aqueous buffer media over a wide frequency range of 4 Hz to 20 GHz.

Electrical impedance spectroscopy was performed on suspensions of plant cells in aqueous buffer media over a wide frequency range of 4 Hz to 20 GHz. Custom probes were designed, manufactured, and used for these investigations. Experiments were performed with a custom-made parallel plate probe and impedance analysers in the low-frequency range (4 Hz to 5 MHz), with a custom-made coaxial airline probe and a vector network analyser in the mid-frequency range (100 kHz to 3 GHz), and with a commercial open-ended probe and a vector network analyser in the high-frequency range (200 MHz to 20 GHz). The impedance data acquired were processed in order to eliminate the effects of parasitics and compensate for geometrical differences between the three probes. Following this, the data were fitted to a unified model consisting of the Randles and Debye models. The data were also normalized to a reference measurement, in order to accentuate the effects of cell concentration on the impedance of the suspensions.•The methodology allows for impedance spectroscopy of cell suspensions over a wide frequency range spanning 10 orders of magnitude.•It allows for compensation of parasitics and of geometrical variations between probes, using mathematical techniques.

EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2.

Plants are constantly being challenged by aspiring pathogens. In order to protect themselves, plants have developed numerous defense mechanisms that are either specific or non-specific to the pathogen. Pattern recognition receptors can trigger plant defense responses in response to specific ligands or patterns. EIX (ethylene-inducing xylanase) triggers a defense response via the LeEix2 receptor, while bacterial flagellin triggers plant innate immunity via the FLS2 receptor. Endocytosis has been suggested to be crucial for the process in both cases. Here we show that the EIX elicitor triggers internalization of the LeEix2 receptor. Treatment with endocytosis, actin or microtubule inhibitors greatly reduced the internalization of LeEix2. Additionally, we demonstrate that plant EHD2 binds to LeEix2 and is an important factor in its internalization and in regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis and induction of pathogenesis-related protein expression in the case of EIX/LeEix2 (an LRR receptor lacking a kinase domain), but does not appear to be involved in the FLS2 system (an LRR receptor possessing a kinase domain). Our results suggest that various endocytosis pathways are involved in the induction of plant defense responses.

Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance.

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant's response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.