Functional characterization of plant specific Indeterminate Domain (IDD) transcription factors in tomato (Solanum lycopersicum L.).

Plant-specific transcription factors (TFs) are responsible for regulating the genes involved in the development of plant-specific organs and response systems for adaptation to terrestrial environments. This includes the development of efficient water transport systems, efficient reproductive organs, and the ability to withstand the effects of terrestrial factors, such as UV radiation, temperature fluctuations, and soil-related stress factors, and evolutionary advantages over land predators. In rice and Arabidopsis, INDETERMINATE DOMAIN (IDD) TFs are plant-specific TFs with crucial functions, such as development, reproduction, and stress response. However, in tomatoes, IDD TFs remain uncharacterized. Here, we examined the presence, distribution, structure, characteristics, and expression patterns of SlIDDs. Database searches, multiple alignments, and motif alignments suggested that 24 TFs were related to Arabidopsis IDDs. 18 IDDs had two characteristic C2H2 domains and two C2HC domains in their coding regions. Expression analyses suggest that some IDDs exhibit multi-stress responsive properties and can respond to specific stress conditions, while others can respond to multiple stress conditions in shoots and roots, either in a tissue-specific or universal manner. Moreover, co-expression database analyses suggested potential interaction partners within IDD family and other proteins. This study functionally characterized SlIDDs, which can be studied using molecular and bioinformatics methods for crop improvement.

Integrated Full-Length Transcriptomics and Metabolomics Reveal Glycosyltransferase Involved in the Biosynthesis of Flavonol Glycosides in Laportea bulbifera.

Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite profiles and associated biosynthetic mechanisms have not been well-studied. Using Laportea bulbifera as a model, we conducted widely targeted metabolomics, which revealed 523 secondary metabolites, including a unique accumulation of flavonol glycosides in bulblet. Through full-length transcriptomic and RNA-seq analyses, the related genes in the flavonoid biosynthesis pathway were identified. Finally, weighted gene correlation network analysis and functional characterization revealed four LbUGTs, including LbUGT78AE1, LbUGT72CT1, LbUGT71BX1, and LbUGT71BX2, can catalyze the glycosylation of flavonol aglycones (kaempferol, myricetin, gossypetin, and quercetagetin) using UDP-Gal and UDP-Glu as the sugar donors. LbUGT78AE1 and LbUGT72CT1 showed substrate promiscuity, whereas LbUGT71BX1 and LbUGT71BX2 exhibited different substrate and sugar donor selectivity. These results provide a genetic resource for studying Laportea in the Urticaceae family, as well as key enzymes responsible for the metabolism of valuable flavonoid glycosides.

Origin, evolution, and diversification of inositol 1,4,5-trisphosphate 3-kinases in plants and animals.

BACKGROUND: In Eukaryotes, inositol polyphosphates (InsPs) represent a large family of secondary messengers and play crucial roes in various cellular processes. InsPs are synthesized through a series of pohophorylation reactions catalyzed by various InsP kinases in a sequential manner. Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K), one member of InsP kinase, plays important regulation roles in InsPs metabolism by specifically phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4) in animal cells. IP3Ks were widespread in fungi, plants and animals. However, its evolutionary history and patterns have not been examined systematically. RESULTS: A total of 104 and 31 IP3K orthologues were identified across 57 plant genomes and 13 animal genomes, respectively. Phylogenetic analyses indicate that IP3K originated in the common ancestor before the divergence of fungi, plants and animals. In most plants and animals, IP3K maintained low-copy numbers suggesting functional conservation during plant and animal evolution. In Brassicaceae and vertebrate, IP3K underwent one and two duplication events, respectively, resulting in multiple gene copies. Whole-genome duplication (WGD) was the main mechanism for IP3K duplications, and the IP3K duplicates have experienced functional divergence. Finally, a hypothetical evolutionary model for the IP3K proteins is proposed based on phylogenetic theory. CONCLUSION: Our study reveals the evolutionary history of IP3K proteins and guides the future functions of animal, plant, and fungal IP3K proteins.

Potato miR394 targets StA/N-INVE and StLCR to negatively regulate late blight resistance.

MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.

Silicon supplementation stabilizes the effect of copper stress, the use of copper chaperones and genes involved: a review.

Heavy metal stress is a major problem in present scenario and the consequences are well known. The agroecosystems are heavily affected by the heavy metal stress and the question arises on the sustainability of the agricultural products. Heavy metals inhibit the process to influence the reactive oxygen species production. When abundantly present copper metal ion has toxic effects which is mitigated by the exogenous application of Si. The role of silicon is to enhance physical parameters as well as gas exchange parameters. Si is likely to increase antioxidant enzymes in response to copper stress which can relocate toxic metals at subcellular level and remove heavy metals from the cell. Silicon regulates phytohormones when excess copper is present. Rate of photosynthesis and mineral absorption is increased in response to metal stress. Silicon manages enzymatic and non-enzymatic activities to balance metal stress condition. Cu transport by the plasma membrane is controlled by a family of proteins called copper transporter present at cell surface. Plants maintain balance in absorption, use and storage for proper copper ion homeostasis. Copper chaperones play vital role in copper ion movement within cells. Prior to that metallochaperones control Cu levels. The genes responsible in copper stress mitigation are discovered in various plant species and their function are decoded. However, detailed molecular mechanism is yet to be studied. This review discusses about the crucial mechanisms of Si-mediated alleviation of copper stress, the role of copper binding proteins in copper homeostasis. Moreover, it also provides a brief information on the genes, their function and regulation of their expression in relevance to Cu abundance in different plant species which will be beneficial for further understanding of the role of silicon in stabilization of copper stress.

Climate, litter quality and radiation duration jointly regulate the net effect of UV radiation on litter decomposition.

Photodegradation via ultraviolet (UV) radiation is an important factor driving plant litter decomposition. Despite increasing attention to the role of UV photodegradation in litter decomposition, the specific impact of UV radiation on the plant litter decomposition stage within biogeochemical cycles remains unclear at regional and global scales. To clarify the variation rules of magnitude of UV effect on plant litter decomposition and their regulatory factors, we conducted a meta-analysis based on 54 published papers. Our results indicated that UV significantly promoted the mass loss of litter by facilitating decay of carbonaceous fractions and release of nitrogen and phosphorus. The promotion effect varied linearly or non-linearly with the time that litter exposed to UV, and with climatic factors. The UV effect on litter decomposition decreased first than increased on precipitation and temperature gradients, reaching its minimum in the area with a precipitation of 400-600 mm, and a temperature of 15-20 °C. This trend might be attributed to a potential equilibrium between the photofacilitation and photo-inhibition effects of UV under this condition. This variation in UV effect on precipitation gradient was in agreement with the fact that UV photodegradation effect was weakest in grassland ecosystems compared to that in forest and desert ecosystems. In addition, initial litter quality significantly influenced the magnitude of UV effect, but had no influence on the correlation between UV effect and climate gradient. Litter with lower initial nitrogen and lignin content shown a greater photodegradation effect, whereas those with higher hemicellulose and cellulose content had a greater photodegradation effect. Our study provides a comprehensive understanding of photodegradation effect on plant litter decomposition, indicates potentially substantial impacts of global enhancements of litter decomposition by UV, and highlights the necessity to quantify the contribution of photochemical minerallization pathway and microbial degradation pathway in litter decomposition.

[Identification and expression of genome of uridine diphosphate glycosyltransferase (UGT) gene family from Chrysanthemum indicum].

Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth and development and regulation of secondary metabolism. Based on the genome of a diploid Chrysanthemum indicum, the UGT gene family from Ch. indicum was identified by bioinformatics methods, and the physical and chemical properties, subcellular localization prediction, conserved motif, phylogeny, chromosome location, gene structure, and gene replication events of UGT protein were analyzed. Transcriptome and real-time fluorescence quantitative polymerase chain reaction(PCR) were used to analyze the expression pattern of the UGT gene in flowers and leaves of Ch. indicum. Quasi-targeted metabolomics was used to analyze the differential metabolites in flowers and leaves. The results showed that a total of 279 UGT genes were identified in the Ch. indicum genome. Phylogenetic analysis showed that these UGT genes were divided into 8 subfamilies. Members of the same subfamily were distributed in clusters on the chromosomes. Tandem duplications were the main driver of the expansion of the UGT gene family from Ch. indicum. Structural domain analysis showed that 262 UGT genes had complete plant secondary metabolism signal sequences(PSPG box). The analysis of cis-acting elements indicated that light-responsive elements were the most ubiquitous elements in the promoter regions of UGT gene family members. Quasi-targeted metabolome analysis of floral and leaf tissue revealed that most of the flavonoid metabolites, including luteolin-7-O-glucoside and kaempferol-7-O-glucoside, had higher accumulation in flowers. Comparative transcriptome analysis of flower and leaf tissue showed that there were 72 differentially expressed UGT genes, of which 29 genes were up-regulated in flowers, and 43 genes were up-regulated in leaves. Correlation network and phylogenetic analysis showed that CindChr9G00614970.1, CindChr2G00092510.1, and CindChr2G00092490.1 may be involved in the synthesis of 7-O-flavonoid glycosides in Ch. indicum, and real-time fluorescence quantitative PCR analysis further confirmed the reliability of transcriptome data. The results of this study are helpful to understand the function of the UGT gene family from Ch. indicum and provide data reference and theoretical basis for further study on the molecular regulation mechanism of flavonoid glycosides synthesis in Ch. indicum.

Selenium in plants: A nexus of growth, antioxidants, and phytohormones.

Selenium (Se) is an essential micronutrient for both human and animals. Plants serve as the primary source of Se in the food chain. Se concentration and availability in plants is influenced by soil properties and environmental conditions. Optimal Se levels promote plant growth and enhance stress tolerance, while excessive Se concentration can result in toxicity. Se enhances plants ROS scavenging ability by promoting antioxidant compound synthesis. The ability of Se to maintain redox balance depends upon ROS compounds, stress conditions and Se application rate. Furthermore, Se-dependent antioxidant compound synthesis is critically reliant on plant macro and micro nutritional status. As these nutrients are fundamental for different co-factors and amino acid synthesis. Additionally, phytohormones also interact with Se to promote plant growth. Hence, utilization of phytohormones and modified crop nutrition can improve Se-dependent crop growth and plant stress tolerance. This review aims to explore the assimilation of Se into plant proteins, its intricate effect on plant redox status, and the specific interactions between Se and phytohormones. Furthermore, we highlight the proposed physiological and genetic mechanisms underlying Se-mediated phytohormone-dependent plant growth modulation and identified research opportunities that could contribute to sustainable agricultural production in the future.

Small but powerful: RALF peptides in plant adaptive and developmental responses.

Plants live in a highly dynamic environment and require to rapidly respond to a plethora of environmental stimuli, so that to maintain their optimal growth and development. A small plant peptide, rapid alkalization factor (RALF), can rapidly increase the pH value of the extracellular matrix in plant cells. RALFs always function with its corresponding receptors. Mechanistically, effective amount of RALF is induced and released at the critical period of plant growth and development or under different external environmental factors. Recent studies also highlighted the role of RALF peptides as important regulators in plant intercellular communications, as well as their operation in signal perception and as ligands for different receptor kinases on the surface of the plasma membrane, to integrate various environmental cues. In this context, understanding the fine-print of above processes may be essential to solve the problems of crop adaptation to various harsh environments under current climate trends scenarios, by genetic means. This paper summarizes the current knowledge about the structure and diversity of RALF peptides and their roles in plant development and response to stresses, highlighting unanswered questions and problems to be solved.

Non-toxic orange carbon dots stimulate photosynthesis and CO2 assimilation in hydroponically cultivated green beans (Phaseolus vulgaris).

Continuous increasing leaf photosynthesis may enhance plant yield. As an evolutionary property, plants use less photosynthetic capacity than is theoretically possible. Plant nanobionics is a bioengineering field that improves plant functions using nanoparticles. We applied orange carbon dots (o-CDs) onto the foliage of green beans (Phaseolus vulgaris ) grown in hydroponics to improve their photosynthetic performance and CO2 assimilation. Photosynthesis parameters, photosynthetic pigments content, total phenolic content (TPC) and antioxidative activity (TAA) were measured. Results show that photosynthetic pigments remained unchanged, while photosynthesis was improved. Both o-CDs concentrations decreased TPC and TAA. The light response curve showed higher CO2 assimilation at both o-CDs concentrations, particularly at lower light intensity. Correlation analysis confirmed increased CO2 binding and assimilation at 1mg L-1 . This study demonstrated the potential of using o-CDs as a safe biostimulator through photosynthesis increase and CO2 assimilation without toxic effects on plants. This may stimulate yield increase that paves the way for their agricultural application.

Metabolomics-driven investigation of plant defense response against pest and pathogen attack.

The advancement of metabolomics has assisted in the identification of various bewildering characteristics of the biological system. Metabolomics is a standard approach, facilitating crucial aspects of system biology with absolute quantification of metabolites using minimum samples, based on liquid/gas chromatography, mass spectrometry and nuclear magnetic resonance. The metabolome profiling has narrowed the wide gaps of missing information and has enhanced the understanding of a wide spectrum of plant-environment interactions by highlighting the complex pathways regulating biochemical reactions and cellular physiology under a particular set of conditions. This high throughput technique also plays a prominent role in combined analyses of plant metabolomics and other omics datasets. Plant metabolomics has opened a wide paradigm of opportunities for developing stress-tolerant plants, ensuring better food quality and quantity. However, despite advantageous methods and databases, the technique has a few limitations, such as ineffective 3D capturing of metabolites, low comprehensiveness, and lack of cell-based sampling. In the future, an expansion of plant-pathogen and plant-pest response towards the metabolite architecture is necessary to understand the intricacies of plant defence against invaders, elucidation of metabolic pathway operational during defence and developing a direct correlation between metabolites and biotic stresses. Our aim is to provide an overview of metabolomics and its utilities for the identification of biomarkers or key metabolites associated with biotic stress, devising improved diagnostic methods to efficiently assess pest and pathogen attack and generating improved crop varieties with the help of combined application of analytical and molecular tools.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site.

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we study glycoside hydrolase family 19 (GH19) chitinase-an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discover that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrate that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.

Divergence in regulatory mechanisms of GR-RBP genes in different plants under abiotic stress.

The IVa subfamily of glycine-rich proteins (GRPs) comprises a group of glycine-rich RNA binding proteins referred to as GR-RBPa here. Previous studies have demonstrated functions of GR-RBPa proteins in regulating stress response in plants. However, the mechanisms responsible for the differential regulatory functions of GR-RBPa proteins in different plant species have not been fully elucidated. In this study, we identified and comprehensively studied a total of 34 GR-RBPa proteins from five plant species. Our analysis revealed that GR-RBPa proteins were further classified into two branches, with proteins in branch I being relatively more conserved than those in branch II. When subjected to identical stresses, these genes exhibited intensive and differential expression regulation in different plant species, corresponding to the enrichment of cis-acting regulatory elements involving in environmental and internal signaling in these genes. Unexpectedly, all GR-RBPa genes in branch I underwent intensive alternative splicing (AS) regulation, while almost all genes in branch II were only constitutively spliced, despite having more introns. This study highlights the complex and divergent regulations of a group of conserved RNA binding proteins in different plants when exposed to identical stress conditions. These species-specific regulations may have implications for stress responses and adaptations in different plant species.

Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Phthalate acid esters: A review of aquatic environmental occurrence and their interactions with plants.

The increasing use of phthalate acid esters (PAEs) in various applications has inevitably led to their widespread presence in the aquatic environment. This presents a considerable threat to plants. However, the interactions between PAEs and plants in the aquatic environment have not yet been comprehensively reviewed. In this review, the properties, occurrence, uptake, transformation, and toxic effects of PAEs on plants in the aquatic environment are summarized. PAEs have been prevalently detected in the aquatic environment, including surface water, groundwater, seawater, and sediment, with concentrations ranging from the ng/L or ng/kg to the mg/L or mg/kg range. PAEs in the aquatic environment can be uptake, translocated, and metabolized by plants. Exposure to PAEs induces multiple adverse effects in aquatic plants, including growth perturbation, structural damage, disruption of photosynthesis, oxidative damage, and potential genotoxicity. High-throughput omics techniques further reveal the underlying toxicity molecular mechanisms of how PAEs disrupt plants on the transcription, protein, and metabolism levels. Finally, this review proposes that future studies should evaluate the interactions between plants and PAEs with a focus on long-term exposure to environmental PAE concentrations, the effects of PAE alternatives, and human health risks via the intake of plant-based foods.

Leaf chlorophyll content is the crucial factor for the temporal and spatial variation of global plants leaf maximum carboxylation rate.

Photosynthesis plays an important role in the terrestrial carbon and water cycles which are often studied using terrestrial biosphere models (TBMs). The maximum carboxylation rate at 25 °C (Vcmax25) is a key parameter in the photosynthesis module of TBMs, yet the spatiotemporal distribution of Vcmax25 and the driving mechanism are not fully understood. In this study, Enzyme Kinetics response model, leaf chlorophyll content response model and partial correlation analysis were used to analyze the temporal and spatial changes patterns of atmospheric environment, enzyme dynamic and soil nutrition on Vcmax25 and the driving mechanism, and has made a few useful conclusions: (1) Vcmax25 varies significantly with latitude and between- and within-plant function types (PFTs), which mainly dependent on leaf chlorophyll content (LCC). Under the influence of temperature, the contribution of LCC to the seasonal variation of Vcmax25 is very different among the eight main biomes, with an average contribution of 21 %. (2) The relationship between meteorological variables and Vcmax25 was significant, due to the fact that meteorological variables drive the Rubisco enzyme content that have a significant relationship with Vcmax25, rather than directly acting on Vcmax25. (3) Soil nutrient elements had significant influence on the spatiotemporal variation of Vcmax25 and LCC. The results showed that soil total carbon, soil nitrogen and organic carbon not only affect the temporal and spatial pattern of Vcmax25, but also are the key factors of LCC temporal-spatial variation. These findings provide useful information for better parameterization of Vcmax25 in TBMs.

Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover.

MAIN CONCLUSION: Carbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field. Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

Assessing the High Temperature Effects on Stomatal Production.

The production of stomata, the epidermal pores of plants, is influenced by diverse environmental signals including high temperature. To assess its impact on stomatal formation, researchers need to grow plants in a carefully designed regime under controlled conditions and capture clear, microscopic views of the epidermis. Here, we describe a procedure to study the effect of high temperature on stomatal formation. This method can generate high-quality epidermal images of cotyledons, leaves, and hypocotyl of young Arabidopsis seedlings, which allow the determination of the pattern, density, and index of stomata on these tissues. Besides temperature, the protocol can serve as a general approach to examine stomatal phenotype and the effect of other external signals on stomatal formation.

Investigating the Effects of Temperature on Pathogen Propagation in Arabidopsis.

Temperature is one of the most prominent environmental factors that influence plant immunity. Depending on the plant-pathogen system, increased temperature may inhibit or enhance disease resistance or immunity in plants. Measuring the effect of temperature on plant immunity is the first step toward revealing climate effects on plant-pathogen interactions and molecular regulators of temperature sensitivity of plant immunity. Quantification of plant disease resistance or susceptibility under different temperatures can be accomplished by assessing pathogen growth over time in infected plants or tissues. Here, we present a protocol for quantifying pathogen growth in the most studied system of Arabidopsis thaliana and Pseudomonas syringae pathovar tomato (Pst) DC3000. We discuss important factors to consider for assaying pathogen growth in plants under different temperatures. This protocol can be used to assess temperature sensitivity of resistance in different plant genotypes and to various pathovars.