Genome-Wide Identification of the WRKY Gene Family and Functional Characterization of CpWRKY5 in Cucurbita pepo.

The WRKY gene family is crucial for regulating plant growth and development. However, the WRKY gene is rarely studied in naked kernel formation in hull-less Cucurbita pepo L. (HLCP), a natural mutant that lacks the seed coat. In this research, 76 WRKY genes were identified through bioinformatics-based methods in C. pepo, and their phylogenetics, conserved motifs, synteny, collinearity, and temporal expression during seed coat development were analyzed. The results showed that 76 CpWRKYs were identified and categorized into three main groups (I-III), with Group II further divided into five subgroups (IIa-IIe). Moreover, 31 segmental duplication events were identified in 49 CpWRKY genes. A synteny analysis revealed that C. pepo shared more collinear regions with cucumber than with melon. Furthermore, quantitative RT-PCR (qRT-PCR) results indicated the differential expression of CpWRKYs across different varieties, with notable variations in seed coat development between HLCP and CP being attributed to differences in CpWRKY5 expression. To investigate this further, CpWRKY5-overexpression tobacco plants were generated, resulting in increased lignin content and an upregulation of related genes, as confirmed by qRT-PCR. This study offers valuable insights for future functional investigations of CpWRKY genes and presents novel information for understanding the regulation mechanism of lignin synthesis.

GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

Trichoderma longibrachiatum T6: A nematocidal activity of endochitinase gene exploration and its function identification.

Endochitinase is a natural extracellular protein in Trichoderma longibrachiatum T6, which can degrade the eggshell of Heterodera avenae significantly, however the related genes that coding this protein was rarely characterized. In the present study, the endochitinase 18-5 gene (T6-Echi18-5) of T. longibrachiatum T6 was cloned and sequenced. The expression level of T6-Echi18-5 gene in T. longibrachiatum T6 was induced and increased after the H. avenae cysts inoculation. The full-length cDNA sequence of T6-Echi18-5 was 1671 bp that contained an ORF of 1275 bp, corresponding to 424 amino acids with a 45.9 kDa molecular weight. A single band of 60.04 kDa was detected and identified using SDS-PAGE and Western blot analysis after transferring the T6-Echi18-5 gene to Escherichia coli BL21 Rosetta (DE3). The concentration of purified recombinant T6-Echi18-5 protein was 1.53 mg·ml-1, and the optimal temperature and pH were 50 °C and 5.0, respectively. The eggshell and content were dissolved and exuded from 4 to10 days after treatment with the purified recombinant T6-Echi18-5 protein. The relative inhibition rate of eggs hatching was 86.79 % at 12 days after treatment. Our study demonstrated the key role of T6-Echi18-5 gene in degrading the H. avenae eggshell and inhibiting the eggs hatching.

JAZ8 Interacts With VirE3 Attenuating Agrobacterium Mediated Root Tumorigenesis.

Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.

Optimization of the Fermentation Media and Parameters for the Bio-control Potential of Trichoderma longibrachiatum T6 Against Nematodes.

The cereal cyst nematode Heterodera avenae is one of the important soil-borne pathogens of cereal crops and causes high yield losses worldwide. Trichoderma spp. formulations are applied as commercial bio-control agents against soil-borne plant pathogens such as H. avenae. However, the relationship between Trichoderma longibrachiatum fermentation parameters and its bio-control potential against H. avenae has not been exclusively established. In the present study, the effect of 10 different fermentation media and conditions on the nematicidal activity of T. longibrachiatum T6 (T6) was evaluated with a single-factor method and a Plackett-Burman design, and the interaction between different fermentation parameters was investigated by a Box-Behnken design. The variables for enhancing the nematicidal activity of T6 culture filtrates were explored and optimized using response surface methodology (RSM). The Minor Medium (MM) plus wheat bran-2 medium was found to be the most effective fermentation medium for T6 culture filtrates against the second stage juveniles (J2s) of H. avenae. The maximum mortality of the J2s was obtained using the T6 culture filtrates under the following fermentation conditions: initial pH 6, 28°C culture temperature, 180 rpm rotating speed, 60 ml of fermentation media, 7 days of incubation time, and 1 ml of inoculation volumes. Among these parameters, the initial pH, inoculation volume, and incubation day were identified as the most significant parameters and critical independent variables for enhancing the nematicidal activity of the T6 culture filtrates. After further optimizations based on statistical predictions, the highest nematicidal activity (92.42%) was obtained with the T6 culture filtrates fermented under an initial pH of 6.06, an inoculation volume of 1.62 ml, and an incubation time of 7.15 days. The nematicidal activity was increased approximately by as high as 1.07% compared with that before optimization. Bio-control efficacy of T6 culture filtrates was 83.88% at the 70th day after wheat seeds sowing in greenhouse experiments. The results from the validation experiments agreed with the model predictions. Our study has improved the bio-control potential of Trichoderma spp. against the plant-parasitic nematodes H. avenae and provided a cost-efficient bio-resource in the future development of novel bio-control agents.

Mechanisms and Characterization of Trichoderma longibrachiatum T6 in Suppressing Nematodes (Heterodera avenae) in Wheat.

Heterodera avenae is an important soil-borne pathogen that affects field crops worldwide. Chemical nematicides can be used to control the nematode, but they bring toxicity to the environment and human. Trichoderma longibrachiatum has been shown to have the ability to control H. avenae cysts, but detailed microscopic observations and bioassays are lacking. In this study, we used microscopic observations and bioassays to study the effect of T. longibrachiatum T6 (TL6) on the eggs and second stage juveniles (J2s) of H. avenae, and investigate the role of TL6 in inducing the resistance to H. avenae in wheat seedling at physiological and biochemical levels. Microscopic observations recorded that TL6 parasitized on the H. avenae eggs, germinated, and produced a large number of hyphae on the eggs surface at the initial stage, thereafter, the eggs were completely surrounded by dense mycelia and the contents of eggs were lysed at the late stage. Meanwhile, the conidia suspension of TL6 parasitized on the surface of J2s, produced a large number of hyphae that penetrated the cuticle and caused deformation of the nematodes. TL6 at the concentration of 1.5 × 107 conidia ml-1 had the highest rates of parasitism on eggs and J2s, reflected by the highest hatching-inhibition of eggs and the mortality of J2s. In the greenhouse experiments, wheat seedlings treated with TL6 at 1.5 × 107 conidia ml-1 had reduced H. avenae infection, and increased plant growth significantly compared to the control. The cysts and juveniles in soil were reduced by 89.8 and 92.7%, the juveniles and females in roots were reduced by 88.3 and 91.3%, whereas the activity of chitinase and ß-1, 3-glucanase, total flavonoids and lignin contents in wheat roots were increased significantly at different stage after inoculation with the eggs and TL6 conidia in comparison to the control. Maximum activity of chitinase and ß-1, 3-glucanase were recorded at the 20th and 15th Days after inoculation with TL6 and thereafter it declined. The maximum contents of total flavonoids and lignin were recorded at the 35th and 40th Days after inoculation with TL6. After being stained with the rapid vital dyes of acridine orange (AO) and neutral red (NR), the frozen and infected eggs and J2s of H. avenae changed color to orange and red, respectively, while the color of eggs and J2s in control group did not change. Therefore, our results suggest that TL6 is potentially an effective bio-control agent for H. avenae. The possible mechanisms by which TL6 suppresses H. avenae infection are due to the direct parasitic and lethal effect of TL6 on the eggs and J2s activity, and the induced defense response in wheat plants together.

[Parasitic and lethal action of Trichoderma longibrachiatum against Heterodera avenae].

OBJECTIVE: To evaluate the potential of Trichoderma longibrachiatum spore suspension against Heterodera avenae. METHODS: The parasitic and lethal effects of T. longibrachiatum spore suspension against the cysts of H. avenae were studied in vitro and observed under microscope. RESULTS: Microscopic observation showed that the spore suspension of T. longibrachiatum parasitized on the cyst surface, germinated a large number of hyphae, and grew on the surface of the cyst at the initial stage. Later, the cysts were completely surrounded by dense mycelium, and the contents of digestion in cysts was lysed, even some cysts produced vacuoles, and some were split up and finally the cyst was dissolved by the metabolite of T. longibrachiatum. In vitro studies showed that high concentrations of T. longibrachiatum spores had strong parasitic and lethal effects on the cysts of H. avenae, and the probable mechanism of parasitic and lethal effects of T. longibrachiatum against H. avenae were mainly by inducing and increasing chitinase, glucanase and caseinase activity. The cysts were parasitized by 93.3% at 18 days, the hatching of cysts were inhibited by 93.6% at 10 days when treated with the concentrations (1.5 x 10(8) CFU/mL) of T. longibrachiatum. CONCLUSION: Trichoderma longibrachiatum had strong parasitic and lethal effects on the cysts of H. avenae, and has the potential as a new biocontrol agent.

[Parasitic and lethal effects of Trichoderma longibrachiatum on Heterodera avenae: microscopic observation and bioassay].

A laboratory experiment was conducted to study the parasitic and lethal effects of Trichoderma longibrachiatum conidia suspension on Heterodera avenae cysts. Different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum conidia suspension had strong parasitic and lethal effects on H. avenae cysts, and the effects differed significantly among the different concentrations. When treated with the T. longibrachiatum conidia suspension at a concentration of 1.5 x 10(7) cfu x mL(-1), 96.7% of the H. avenae cysts were parasitized by the conidia at the 18th day, and the hatching rate of the cysts was inhibited by 91.2% at the 22nd day. The microscopic observation showed that at the initial parasitic stage, T. longibrachiatum conidia suspension adhered or parasitized on the cyst surface, germinated a large number of hyphae, and grew on the cyst surface, making the development of cyst embryo stopped and the contents in cysts flocculated, and even, some cysts started to deform, and small dark brown vacuoles formed on the cyst surface. At the later parasitic stage, the cysts were penetrated by dense mycelium, cysts were broken, their contents exosmosed, and the mycelium on the integument of some cysts produced conidiophores, on which, conidium were adhered or parasitized. It was considered that T. longibrachiatum could be used as a potential high-efficient bioagent to control the occurrence and damage of H. avenae.