Cucumber (Cucumis sativus L.) translationally controlled tumor protein interacts with CsRab11A and promotes activation of target of rapamycin in response to Podosphaera xanthii.

The target of rapamycin (TOR) kinase serves as a central regulator that integrates nutrient and energy signals to orchestrate cellular and organismal physiology in both animals and plants. Despite significant advancements having been made in understanding the molecular and cellular functions of plant TOR kinases, the upstream regulators that modulate TOR activity are not yet fully elucidated. In animals, the translationally controlled tumor protein (TCTP) is recognized as a key player in TOR signaling. This study reveals that two TCTP isoforms from Cucumis sativus, when introduced into Arabidopsis, are instrumental in balancing growth and defense mechanisms against the fungal pathogen Golovinomyces cichoracearum. We hypothesize that plant TCTPs act as upstream regulators of TOR in response to powdery mildew caused by Podosphaera xanthii in Cucumis. Our research further uncovers a stable interaction between CsTCTP and a small GTPase, CsRab11A. Transient transformation assays indicate that CsRab11A is involved in the defense against P. xanthii and promotes the activation of TOR signaling through CsTCTP. Moreover, our findings demonstrate that the critical role of TOR in plant disease resistance is contingent upon its regulated activity; pretreatment with a TOR inhibitor (AZD-8055) enhances cucumber plant resistance to P. xanthii, while pretreatment with a TOR activator (MHY-1485) increases susceptibility. These results suggest a sophisticated adaptive response mechanism in which upstream regulators, CsTCTP and CsRab11A, coordinate to modulate TOR function in response to P. xanthii, highlighting a novel aspect of plant-pathogen interactions.

Cucumis sativus CsbZIP90 suppresses Podosphaera xanthii resistance by modulating reactive oxygen species.

Resistance to disease in plants requires the coordinated action of multiple functionally related genes, as it is difficult to improve disease resistance with a single functional gene. Therefore, the use of transcription factors to regulate the expression of multiple resistance genes to improve disease resistance has become a recent focus in the field of gene research. The basic leucine zipper (bZIP) transcription factor family plays vital regulatory roles in processes, such as plant growth and development and the stress response. In our previous study, CsbZIP90 (Cucsa.134370) was involved in the defense response of cucumber to Podosphaera xanthii, but the relationship between cucumber and resistance to powdery mildew remained unclear. Herein, we detected the function of CsbZIP90 in response to P. xanthii. CsbZIP90 was localized to the cytoplasm and nucleus, and its expression was significantly induced during P. xanthii attack. Transient overexpression of CsbZIP90 in cucumber cotyledons resulted in decreased resistance to P. xanthii, while silencing CsbZIP90 increased resistance to P. xanthii. CsbZIP90 negatively regulated the expression of reactive oxygen species (ROS)-related genes and activities of ROS-related kinases. Taken together, our results show that CsbZIP90 suppresses P. xanthi resistance by modulating ROS. This study will provide target genes for breeding cucumbers resistant to P. xanthii.

Target of rapamycin (TOR) plays a role in regulating ROS-induced chloroplast damage during cucumber (Cucumis sativus) leaf senescence.

In cucumber production, delaying leaf senescence is crucial for improving cucumber yield and quality. Target of rapamycin (TOR) is a highly conserved serine/threonine protein kinase in eukaryotes, which can integrate exogenous and endogenous signals (such as cell energy state levels) to stimulate cell growth, proliferation, and differentiation. However, no studies have yet examined the regulatory role of TOR signalling in cucumber leaf senescence. In this study, the effects of TOR signalling on dark-induced cucumber leaf senescence were investigated using the TOR activator MHY1485 and inhibitor AZD8055 combined with transient transformation techniques. The results indicate that TOR responds to dark-induced leaf senescence, and alterations in TOR activity/expression influence cucumber leaf resistance to dark-induced senescence. Specifically, in plants with elevated TOR activity/expression, we observed reduced expression of senescence-related genes, less membrane lipid damage, decreased cell apoptosis, lower levels of reactive oxygen species production, and less damage to the photosynthetic system compared to the control. In contrast, in plants with reduced TOR activity/expression, we observed higher expression of senescence-related genes, increased membrane lipid damage, enhanced cell apoptosis, elevated levels of reactive oxygen species production, and more damage to the photosynthetic system. These comprehensive results underscore the critical role of TOR in regulating dark-induced cucumber leaf senescence. These findings provide a foundation for controlling premature leaf senescence in cucumber production and offer insights for further exploration of leaf senescence mechanisms and the development of more effective control methods.

Characterization of caffeoyl shikimate esterase gene family identifies CsCSE5 as a positive regulator of Podosphaera xanthii and Corynespora cassiicola pathogen resistance in cucumber.

KEY MESSAGE: CsCSE genes might be involved in the tolerance of cucumber to pathogens. Silencing of the CsCSE5 gene resulted in attenuated resistance of cucumber to Podosphaera xanthii and Corynespora cassiicola. Caffeoyl shikimate esterase (CSE), a key enzyme in the lignin biosynthetic pathway, has recently been characterized to play a key role in defense against pathogenic infection in plants. However, a systematic analysis of the CSE gene family in cucumber (Cucumis sativus) has not yet been conducted. Here, we identified eight CsCSE genes from the cucumber genome via bioinformatic analyses, and these genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Results from multiple sequence alignment indicated that the CsCSE proteins had domains required for CSE activity. Phylogenetic analysis of gene structure and protein motifs revealed the conservation and diversity of the CsCSE gene family. Collinearity analysis showed that CsCSE genes had high homology with CSE genes in wax gourd (Benincasa hispida). Cis-acting element analysis of the promoters suggested that CsCSE genes might play important roles in growth, development, and stress tolerance. Expression pattern analysis indicated that CsCSE5 might be involved in regulating the resistance of cucumber to pathogens. Functional verification data confirmed that CsCSE5 positively regulates the resistance of cucumber to powdery mildew pathogen Podosphaera xanthii and target leaf spot pathogen Corynespora cassiicola. The results of our study provide information that will aid the genetic improvement of resistant cucumber varieties.

The pivotal role of MYB transcription factors in plant disease resistance.

MAIN CONCLUSION: MYB transcription factors are essential for diverse biology processes in plants. This review has focused on the potential molecular actions of MYB transcription factors in plant immunity. Plants possess a variety of molecules to defend against disease. Transcription factors (TFs) serve as gene connections in the regulatory networks controlling plant growth and defense against various stressors. As one of the largest TF families in plants, MYB TFs coordinate molecular players that modulate plant defense resistance. However, the molecular action of MYB TFs in plant disease resistance lacks a systematic analysis and summary. Here, we describe the structure and function of the MYB family in the plant immune response. Functional characterization revealed that MYB TFs often function either as positive or negative modulators towards different biotic stressors. Moreover, the MYB TF resistance mechanisms are diverse. The potential molecular actions of MYB TFs are being analyzed to uncover functions by controlling the expression of resistance genes, lignin/flavonoids/cuticular wax biosynthesis, polysaccharide signaling, hormone defense signaling, and the hypersensitivity response. MYB TFs have a variety of regulatory modes that fulfill pivotal roles in plant immunity. MYB TFs regulate the expression of multiple defense genes and are, therefore, important for increasing plant disease resistance and promoting agricultural production.

The Role and Mechanism of Hydrogen-Rich Water in the Cucumis sativus Response to Chilling Stress.

Cucumber is a warm climate vegetable that is sensitive to chilling reactions. Chilling can occur at any period of cucumber growth and development and seriously affects the yield and quality of cucumber. Hydrogen (H2) is a type of antioxidant that plays a critical role in plant development and the response to stress. Hydrogen-rich water (HRW) is the main way to use exogenous hydrogen. This study explored the role and mechanism of HRW in the cucumber defense response to chilling stress. The research results showed that applying 50% saturated HRW to the roots of cucumber seedlings relieved the damage caused by chilling stress. The growth and development indicators, such as plant height, stem diameter, leaf area, dry weight, fresh weight, and root length, increased under the HRW treatment. Photosynthetic efficiency, chlorophyll content, and Fv/Fm also improved and reduced energy dissipation. In addition, after HRW treatment, the REC and MDA content were decreased, and membrane lipid damage was reduced. NBT and DAB staining results showed that the color was lighter, and the area was smaller under HRW treatment. Additionally, the contents of O2- and H2O2 also decreased. Under chilling stress, the application of HRW increased the activity of the antioxidases SOD, CAT, POD, GR, and APX and improved the expression of the SOD, CAT, POD, GR, and APX antioxidase genes. The GSSG content was reduced, and the GSH content was increased. In addition, the ASA content also increased. Therefore, exogenous HRW is an effective measure for cucumber to respond to chilling stress.

SnRK1 signaling regulates cucumber growth and resistance to Corynespora cassiicola.

Energy metabolism is one of the key factors determining the growth and development of plants and the response to biotic and abiotic stresses. Sucrose non-fermentation 1 related protein kinase 1 (SnRK1) is an important energy-sensitive regulator that plays a key role in the overall control of carbohydrate metabolism. However, little is known about the function of SnRK1 in cucumber. In this study, metformin (an SnRK1 activator) and trehalose (an SnRK1 inhibitor) were used to investigate the role of SnRK1 signaling in cucumber. The results showed that SnRK1 activation could inhibit the growth of cucumber, slow down the net photosynthetic rate (Pn), reduce the contents of photosynthetic pigments and soluble sugars, and suppress the expression of genes related to sucrose metabolism. By contrast, SnRK1 inhibition yielded opposite results. Furthermore, SnRK1 activation and CsSnRK1 over-expression improved cucumber resistance to Corynespora cassiicola. While, SnRK1 inhibition and CsSnRK1 silencing reduced the resistance of cucumber to C. cassiicola. The results indicated that CsSnRK1 gene can positively regulate the resistance of cucumber to C. cassiicola. We conclude that CsSnRK1 signaling plays an important role in balancing the growth and immune response of cucumber. These results can be applied to the improvement of disease-resistant cucumber varieties.

Phosphoproteomics analysis of the effect of target of rapamycin kinase inhibition on Cucumis sativus in response to Podosphaera xanthii.

Target of rapamycin (TOR) kinase is a conserved sensor of cell growth in yeasts, plants, and mammals. Despite the extensive research on the TOR complex in various biological processes, large-scale phosphoproteomics analysis of TOR phosphorylation events upon environmental stress are scarce. Powdery mildew caused by Podosphaera xanthii poses a major threat to the quality and yield of cucumber (Cucumis sativus L.). Previous studies concluded that TOR participated in abiotic and biotic stress responses. Hence, studying the underlying mechanism of TOR-P. xanthii infection is particularly important. In this study, we performed a quantitative phosphoproteomics studies of Cucumis against P. xanthii attack under AZD-8055 (TOR inhibitor) pretreatment. A total of 3384 phosphopeptides were identified from the 1699 phosphoproteins. The Motif-X analysis showed high sensitivity and specificity of serine sites under AZD-8055-treatment or P. xanthii stress, and TOR exhibited a unique preference for proline at +1 position and glycine at -1 position to enhance the phosphorylation response to P. xanthii. The functional analysis suggested that the unique responses were attributed to proteins related to plant hormone signaling, mitogen-activated protein kinase cascade signaling, phosphatidylinositol signaling system, and circadian rhythm; and calcium signaling- and defense response-related proteins. Our results provided rich resources for understanding the molecular mechanism of how the TOR kinase controlled plant growth and stress adaptation.

SlMYC2 interacted with the SlTOR promoter and mediated JA signaling to regulate growth and fruit quality in tomato.

Tomato (Solanum lycopersicum) is a major vegetable crop cultivated worldwide. The regulation of tomato growth and fruit quality has long been a popular research topic. MYC2 is a key regulator of the interaction between jasmonic acid (JA) signaling and other signaling pathways, and MYC2 can integrate the interaction between JA signaling and other hormone signals to regulate plant growth and development. TOR signaling is also an essential regulator of plant growth and development. However, it is unclear whether MYC2 can integrate JA signaling and TOR signaling during growth and development in tomato. Here, MeJA treatment and SlMYC2 overexpression inhibited the growth and development of tomato seedlings and photosynthesis, but increased the sugar-acid ratio and the contents of lycopene, carotenoid, soluble sugar, total phenol and flavonoids, indicating that JA signaling inhibited the growth of tomato seedlings and altered fruit quality. When TOR signaling was inhibited by RAP, the JA content increased, and the growth and photosynthesis of tomato seedlings decreased, indicating that TOR signaling positively regulated the growth and development of tomato seedlings. Further yeast one-hybrid assays showed that SlMYC2 could bind directly to the SlTOR promoter. Based on GUS staining analysis, SlMYC2 regulated the transcription of SlTOR, indicating that SlMYC2 mediated the interaction between JA and TOR signaling by acting on the promoter of SlTOR. This study provides a new strategy and some theoretical basis for tomato breeding.

Lignin biosynthesis regulated by CsCSE1 is required for Cucumis sativus defence to Podosphaera xanthii.

Lignin is a complex phenolic compound that can enhance the stiffness, hydrophobicity, and antioxidant capacity of the cell wall; it thus provides a critical barrier against pathogen and insect invaders. Caffeoyl shikimate esterase (CSE) is a key novel enzyme involved in lignin biosynthesis that is associated with genetic improvements in lignocellulosic biomass; however, no research thus far have revealed the role of CSE in resistance to pathogenic stress. CsCSE1 (Cucsa.134370) has previously been shown to highly associated with the response of cucumber to attack by Podosphaera xanthii through RNA sequencing. Here, we detected the exactly role of CsCSE1 in the defence of cucumber to P. xanthii infection. Homologous sequence alignment revealed that CsCSE1 contains two highly conserved lyase domains (GXSXG), suggesting that CsCSE1 possesses CSE activity. Subcellular localization analysis manifested that CsCSE1 was localized to the plasma membrane and endoplasmic reticulum (ER). Functional analysis demonstrated that the transient silencing of CsCSE1 in cucumber dramatically attenuated resistance to P. xanthii, whereas overexpression of CsCSE1 in cucumber markedly increased resistance to P. xanthii. Further investigation of the abundance of lignin in transient transgenic plants revealed that CsCSE1 might actively mediate the disease resistance of cucumber by promoting lignin biosynthesis. CsCSE1 also affects the expression of its downstream lignin biosynthesis-related genes, like CsLAC, CsCOMT, CsCCR, and CsCAD. The results of this study provide targets for the genetic breeding of tolerant cucumber cultivars as well as new insights that could aid the control of plant diseases.

Transcriptome Sequence Analysis of the Defense Responses of Resistant and Susceptible Cucumber Strains to Podosphaera xanthii.

Powdery mildew (PM) caused by Podosphaera xanthii poses a continuous threat to the performance and yield of the cucumber (Cucumis sativus L.). Control in the initial stages of infection is particularly important. Here, we studied the differential physiological and transcriptomic changes between PM-resistant strain B21-a-2-1-2 and PM-susceptible strain B21-a-2-2-2 at the early stage of P. xanthii attack. When challenged with P. xanthii, the tolerant line can postpone the formation of the pathogen primary germ. Comparative transcriptomic analysis suggested that DEGs related to the cell wall and to pathogen and hormone responses were similar enriched in both cucumber lines under P. xanthii infection. Notably, the number of DEGs triggered by P. xanthii in B21-a-2-1-2 was quintuple that in B21-a-2-2-2, revealing that the success of defense of resistant cucumber is due to rapidly mobilizing multiple responses. The unique responses detected were genes related to SA signaling, MAPK signaling, and Dof and WRKY transcription factors. Furthermore, 5 P. xanthii -inducible hub genes were identified, including GLPK, ILK1, EIN2, BCDHß1, and RGGA, which are considered to be key candidate genes for disease control. This study combined multiple analytical approaches to capture potential molecular players and will provide key resources for developing cucumber cultivars resistant to pathogen stress.

Nucleotide-Binding Leucine-Rich Repeat Genes CsRSF1 and CsRSF2 Are Positive Modulators in the Cucumis sativus Defense Response to Sphaerotheca fuliginea.

Cucumber powdery mildew caused by Sphaerotheca fuliginea is a leaf disease that seriously affects cucumber's yield and quality. This study aimed to report two nucleotide-binding site-leucine-rich repeats (NBS-LRR) genes CsRSF1 and CsRSF2, which participated in regulating the resistance of cucumber to S. fuliginea. The subcellular localization showed that the CsRSF1 protein was localized in the nucleus, cytoplasm, and cell membrane, while the CsRSF2 protein was localized in the cell membrane and cytoplasm. In addition, the transcript levels of CsRSF1 and CsRSF2 were different between resistant and susceptible cultivars after treatment with exogenous substances, such as abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), ethephon (ETH), gibberellin (GA) and hydrogen peroxide (H2O2). The expression analysis showed that the transcript levels of CsRSF1 and CsRSF2 were correlated with plant defense response against S. fuliginea. Moreover, the silencing of CsRSF1 and CsRSF2 impaired host resistance to S. fuliginea, but CsRSF1 and CsRSF2 overexpression improved resistance to S. fuliginea in cucumber. These results showed that CsRSF1 and CsRSF2 genes positively contributed to the resistance of cucumber to S. fuliginea. At the same time, CsRSF1 and CsRSF2 genes could also regulate the expression of defense-related genes. The findings of this study might help enhance the resistance of cucumber to S. fuliginea.

Mildew Resistance Locus O Genes CsMLO1 and CsMLO2 Are Negative Modulators of the Cucumis sativus Defense Response to Corynespora cassiicola.

Corynespora leaf spot caused by Corynespora cassiicola is one of the major diseases in cucumber (Cucumis sativus L.). However, the resistance mechanisms and signals of cucumber to C. cassiicola are unclear. Here, we report that the mildew resistance locus O (MLO) genes, CsMLO1 and CsMLO2, are both negative modulators of the cucumber defense response to C. cassiicola. Subcellular localization analysis showed that CsMLO1 and CsMLO2 are localized in the plasma membrane. Expression analysis indicated that the transcript levels of CsMLO1 and CsMLO2 are linked to the defense response to C. cassiicola. Transient overexpression of either CsMLO1 or CsMLO2 in cucumber cotyledons reduced resistance to C. cassiicola, whereas silencing of either CsMLO1 or CsMLO2 enhanced resistance to C. cassiicola. The relationships of pathogenesis-related proteins, reactive oxygen species (ROS)-associated genes, and abscisic acid (ABA)-related genes to the overexpression and silencing of CsMLO1/CsMLO2 in non-infested cucumber plants were investigated. The results indicated that CsMLO1 mediated resistance against C. cassiicola by regulating the expression of pathogenesis-related proteins and ROS-associated genes, as well as through ABA signaling pathway-associated genes. The CsMLO2-mediated resistance against C. cassiicola primarily involves regulation of the expression of pathogenesis-related proteins. Our findings will guide strategies to enhance the resistance of cucumber to corynespora leaf spot.

The Two Translationally Controlled Tumor Protein Genes, CsTCTP1 and CsTCTP2, Are Negative Modulators in the Cucumis sativus Defense Response to Sphaerotheca fuliginea.

Pathogen stress often significantly decreases cucumber production. However, knowledge regarding the molecular mechanism and signals of cucumber disease resistance is far from complete. Here, we report two translationally controlled tumor protein genes, CsTCTP1 and CsTCTP2, that are both negative modulators in the Cucumis sativus defense response to Sphaerotheca fuliginea. Subcellular localization analysis showed that CsTCTP1 and CsTCTP2 were both localized in the cytoplasm. Expression analysis indicated that the transcript levels of CsTCTP1 and CsTCTP2 were linked to the degree of cucumber resistance to S. fuliginea. Transient overexpression of either CsTCTP1 or CsTCTP2 in cucumber cotyledons impaired resistance to S. fuliginea, whereas silencing of either CsTCTP1 or CsTCTP2 enhanced cucumber resistance to S. fuliginea. The relationship of several defense-related genes and ABA and target of rapamycin (TOR) signaling pathway-related genes to the overexpressing and silencing of CsTCTP1/CsTCTP2 in non-infested cucumber plants was investigated. The results indicated that CsTCTP1 participates in the defense response to S. fuliginea by regulating the expression of certain defense-associated genes and/or ABA signaling pathway-associated genes, and CsTCTP2 participates through regulating the expression of TOR signaling pathway-associated genes. Our findings will guide enhancing the resistance of cucumber to powdery mildew.

A WRKY gene from Tamarix hispida, ThWRKY4, mediates abiotic stress responses by modulating reactive oxygen species and expression of stress-responsive genes.

WRKY transcription factors are involved in various biological processes, such as development, metabolism and responses to stress. However, their exact roles in abiotic stress tolerance are largely unknown. Here, we demonstrated a working model for the function of a WRKY gene (ThWRKY4) from Tamarix hispida in the stress response. ThWRKY4 is highly induced by abscisic acid (ABA), salt and drought in the early period of stress (stress for 3, 6, or 9 h), which can be regulated by ABF (ABRE binding factors) and Dof (DNA binding with one finger), and also can be crossregulated by other WRKYs and autoregulated as well. Overexpression of ThWRKY4 conferred tolerance to salt, oxidative and ABA treatment in transgenic plants. ThWRKY4 can improve the tolerance to salt and ABA treatment by improving activities of superoxide dismutase and peroxidase, decreasing levels of O2 (-) and H2O2, reducing electrolyte leakage, keeping the loss of chlorophyll, and protecting cells from death. Microarray analyses showed that overexpression of ThWRKY4 in Arabidopsis leads to 165 and 100 genes significantly up- and downregulated, respectively. Promoter scanning analysis revealed that ThWRKY4 regulates the gene expression via binding to W-box motifs present in their promoter regions. This study shows that ThWRKY4 functions as a transcription factor to positively modulate abiotic stress tolerances, and is involved in modulating reactive oxygen species.

A versatile Agrobacterium-mediated transient gene expression system for herbaceous plants and trees.

Plant transient expression is a powerful method used widely for the functional characterization of genes and protein production. In comparison with stable transformation, it has the advantages of being simple, quick, economical, and effective. In the present study, we developed a novel transient gene expression system based on Agrobacterium-mediated transformation. This system is simple and convenient and allows for high transient expression levels. Hyperosmotic pretreatment of plants significantly improved the transient expression in this system. Furthermore, other factors, including acetosyringone concentration, cocultivation time, and Agrobacterium cell density, significantly influenced transient expression efficiency. The results showed that this method is suitable for use with herbaceous plants (such as tobacco and Arabidopsis) and trees (such as birch, poplar, tamarisk, cork, willow, and aralia), suggesting that it may be applied widely in plant transient expression studies.

[Population structure and understory species diversity of different aged Pinus sylvestris var. mongolica plantations in Nenjiang Sandy Land of Northeast China].

Taking the Pinus sylvestris var. mongolica sand-fixing plantations at different development stages (24-, 29-, 39-, and 43 years old) in Nenjiang Sandy Land as test objects, this paper studied their population structure, understory species composition, and species diversity. No regenerated seedlings were found in all the four P. sylvestris var. mongolica plantations. The mean individual height and DBH of the populations differed significantly with development stage. With the increasing age of the plantations, the proportion of small-sized individuals decreased obviously, while that of large-sized individuals increased, population tended to mature, and the diameter structure except 43 years old plantation was in normal distribution. A total of 33 understory plant species were recorded, belonging to 28 genera and 15 families. Setaria viridis was the dominant species, but its dominance decreased gradually with increasing age of the plantations. With the increase of plantation age, the proportion of annual plants decreased, while that of perennial plants increased. The Simpson index and Pielou index had no significant differences among the different aged plantations, but the richness index, Shannon index, and Alatalo index of 39 years old plantation were significantly higher than those of 24 years old stands, suggesting that the species diversity of the community improved with time.