The RLCK subfamily VII-4 controls pattern-triggered immunity and basal resistance to bacterial and fungal pathogens in rice.

Receptor-like cytoplasmic kinases (RLCKs) mediate the intracellular signaling downstream of pattern-recognition receptors (PRRs). Several RLCKs from subfamily VII of rice (Oryza sativa) have important roles in plant immunity, but the role of RLCK VII-4 in pattern-triggered immune (PTI) signaling and resistance to pathogens has not yet been investigated. Here, we generated by multiplex clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing rice sextuple mutant lines where the entire RLCK VII-4 subfamily is inactivated and then analyzed the resulting lines for their response to chitin and flg22 and for their immunity to Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae. Analysis of the rlckvii-4 mutants revealed that they have an impaired reactive oxygen system burst and reduced defense gene expression in response to flg22 and chitin. This indicates that members of the rice RLCK VII-4 subfamily are required for immune signaling downstream of multiple PRRs. Furthermore, we found that the rice RLCK VII-4 subfamily is important for chitin-induced callose deposition and mitogen-activated protein kinase activation and that it is crucial for basal resistance against Xoo and M. oryzae pathogens. This establishes that the RLCK VII-4 subfamily has critical functions in the regulation of multiple PTI pathways in rice and opens the way for deciphering the precise role of its members in the control of rice PTI.

Regulatory networks of hormone-involved transcription factors and their downstream pathways during somatic embryogenesis of Arabidopsis thaliana.

Somatic embryogenesis (SE) depends on a variety of developmental pathways that are influenced by several environmental factors. Therefore, it is important to understand the relationship between environmental and genetic factors by identifying the gene networks involved in SE through gene set enrichment analysis (GSEA). For determination of SE effective transcription factors, upstream sequences of core-enriched genes were analyzed. The results indicated that response to hormones is one of the biological pathways activated by the enriched TFs at all stages of somatic embryogenesis and about half of the hormonal pathways were enriched. On the fifth day after 2,4-Dichlorophenoxyacetic acid (2,4-D) treatment, the activity of hormone-affecting genes reached its maximum. At this time, more transcription factors regulated the enriched genes compared to the other stages of somatic embryogenesis. MYBs, AT-HOOKs, and HSFs are the main families of transcription factors which affect core-enriched genes during SE. CCA1, PRR7, and TOC1 and their related genes at the center of protein-protein interaction of SE-key transcription factors, involved in the regulation of the circadian clock. Gene expression analysis of CCA1, PRR7, and TOC1 revealed that the genes involved in circadian clock reached their maximum activity when embryonic cells formed. Also, auxin response elements were identified at the upstream of SE-circadian clock transcription factors, indicating that they might mediate between auxin signaling and SE-related hormonal pathways as well as SE marker genes such as AGL15, BBM, and LECs. Based on these results, it is possible that the cellular circadian rhythm activates various developmental pathways under the influence of auxin signal transduction and their interactions determine the induction of somatic embryogenesis. According to the results of this study, modifying pathways affected by SE-related transcription factors such as circadian rhythm may result in cell reprogramming and increase somatic embryogenesis efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03546-7.

Genome-wide analysis of annexin gene family in Schrenkiella parvula and Eutrema salsugineum suggests their roles in salt stress response.

Annexins (Anns) play an important role in plant development, growth and responses to various stresses. Although Ann genes have been characterized in some plants, their role in adaptation mechanisms and tolerance to environmental stresses have not been studied in extremophile plants. In this study, Ann genes in Schrenkiella parvula and Eutrema salsugineum were identified using a genome-wide method and phylogenetic relationships, subcellular distribution, gene structures, conserved residues and motifs and also promoter prediction have been studied through bioinformatics analysis. We identified ten and eight encoding putative Ann genes in S. parvula and E. salsugineum genome respectively, which were divided into six subfamilies according to phylogenetic relationships. By observing conservation in gene structures and protein motifs we found that the majority of Ann members in two extremophile plants are similar. Furthermore, promoter analysis revealed a greater number of GATA, Dof, bHLH and NAC transcription factor binding sites, as well as ABRE, ABRE3a, ABRE4, MYB and Myc cis-acting elements in compare to Arabidopsis thaliana. To gain additional insight into the putative roles of candidate Ann genes, the expression of SpAnn1, SpAnn2 and SpAnn6 in S. parvula was studied in response to salt stress, which indicated that their expression level in shoot increased. Similarly, salt stress induced expression of EsAnn1, 5 and 7, in roots and EsAnn1, 2 and 5 in leaves of E. salsugineum. Our comparative analysis implies that both halophytes have different regulatory mechanisms compared to A. thaliana and suggest SpAnn2 gene play important roles in mediating salt stress.

Apoplastic Production of Recombinant AntiVEGF Protein Using Plant-Virus Transient Expression Vector.

Targeting of vascular endothelial growth factor (VEGF) using AntiVEGF can be a promising approach for angiogenesis inhibition and cancer therapy. In this study, we direct AntiVEGF recombinant protein accumulation to cucurbit plant apoplast using a suitable signal (Pr1b) sequence. After assembling the target gene construct and cloning into the expression vector, we infected the plants with the resulting pZYMV-AntiVEGF viral vector. Transcription of the target gene was confirmed with RT-PCR assays. The apoplast-targeted AntiVEGF recombinant protein was detected in infected plants by Dot-blot, western blot, and ELISA analysis. AntiVEGF protein accumulation in the apoplast resulted in levels of 1.2% of TSP (Total Soluble Protein) that demonstrated a two-order increase compared to the cytoplasm-targeted protein. After purification of AntiVEGF protein using aqueous two-phase system (ATPS), purified protein was analyzed with MTT assay. Our results reveal that production of biologically active and correctly processed apoplast-targeted AntiVEGF recombinant protein is possible in plant apoplast. The low level of cytoplasm-targeted AntiVEGF recombinant protein might result from the degradation of improperly folded protein.

Aluminium triggers oxidative stress and antioxidant response in the microalgae Scenedesmus sp.

Aluminium (Al) water pollution is an increasing environmental problem and comprehensive analysis of toxic responses of aquatic primary producer organisms is imperative. We characterized the antioxidant response of Scenedesmus sp. microalga to Al-induced oxidative stress. After 72 h of exposure to Al (0, 10, and 100 µM) in a modified Bold Basal Medium (pH 5.0), we observed cell aggregation and alterations in the subcellular structure, strong lipid peroxidation and oxidative stress induction (detected with the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate) in parallel with Al accumulation in cells. At the same time, Al toxicity caused depletion of important macronutrients like Ca, which is important for cell-wall structure. Analysis of antioxidant enzymatic activities in Al-treated Scenedesmus cells revealed that catalase, ascorbate peroxidase, as well as different isoforms of superoxide dismutase were inhibited especially at the highest Al dose (100 µM), cells that accumulated the highest concentration of Al. On the other hand, glutathione reductase activity increased at that Al concentration. Immunodetection after Western-blotting confirmed that only ascorbate peroxidase inhibition was apparently due to a decrease in enzyme levels. However, the inhibition of catalase and activation of glutathione reductase activities seemed related with post-translational modifications in protein function as protein expression decreased or increased, respectively under Al stress. Our results may help to understand toxic mechanisms triggered by Al in freshwater microalgae, which in turn could aid to select suitable biomarkers of Al contamination in aquatic ecosystems.

Transient expression analysis of synthetic promoters containing F and D cis-acting elements in response to Ascochyta rabiei and two plant defense hormones.

Introduction of a foreign gene coding for a pathogen resistant protein into the target plant and constitutive expression of Resistance (R) proteins may confer high level of resistance. However, genetic engineering could lead to reprogramming of molecular mechanisms that manage physiological behavior, which in turn could lead to undesired results. Therefore, using a pathogen-inducible synthetic promoter approach, response to pathogens could be more specific. Ascochyta rabiei is a destructive fungal pathogen in chickpea production. In this study, we analyzed the expression pattern of three synthetic promoters in response to pathogen and two defense hormones. We have tested three synthetic pathogen-inducible promoters designated as (1) synthetic promoter-D box-D box (SP-DD), (2) synthetic promoter-F element-F element (SP-FF) and (3) synthetic promoter-F element-F element-D box-D box (SP-FFDD) via Agrobacterium transient expression assay. The cis-acting element designated as 'D' is a 31 base pair sequence from the promoter of parsley pathogenesis-related gene 2 (PR2 gene) and the cis-acting element designated as 'F' is a 39 base pairs sequence from the promoter of Arabidopsis AtCMPG1 gene. We used mycelial extracts from two pathotypes of A. rabiei as elicitor to define the responsiveness of the promoters against pathogen. Plant phytohormones including salicylic acid and methyl jasmonate were also used to study the promoter sensitivity in plant signaling pathways. Our results showed that the SP-FF promoter was highly inducible to A. rabiei and methyl jasmonate as well, while the SP-DD promoter was more sensitive to salicylic acid. The SP-FFDD promoter was equally responsive to both pathotypes of A. rabiei which is probably due to the complex nature of box D cis-acting element.

Induction of Immune Response in Animal Model Using Recombinant Anti-NDV Vaccine.

BACKGROUND: Newcastle disease is a major avian disease that causes enormous economic loss in poultry industry. There have been a number of reports on the suitability of plant-based recombinant vaccine against this disease. Fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) represent the major immunogenic sites for development of recombinant anti-ND vaccines in plant hosts. OBJECTIVES: The main objective of this research was to evaluate the ability of a recombinant anti-ND vaccine in induction of immune responses in animal model. MATERIALS AND METHODS: In this study, immunogenicity of recombinant fusion (F) and hemagglutinin-neuraminidase (HN) epitopes of the Newcastle disease virus (NDV) is investigated in an animal model. The corresponding genes encoding amino acids 65-81 of the F protein and 346-353 amino acids of HN were expressed in tobacco seedling using agrobacterium-mediated transformation. Expression of the foreign gene in the tobacco seedlings was investigated by a number of molecular assays including Real-Time PCR and ELISA. Transgenic plant extract was used to induce immunogenic response in animal model. RESULTS: Integration of the foreign gene in plant host genome was confirmed by polymerase chain reaction (PCR). Expression of the foreign recombinant protein was confirmed by Real-Time PCR and ELISA assays. Immunogenicity of the recombinant protein was investigated in rabbit by subcutaneous injection. Results indicated that the transgenic plant extract can induce immune responses in the host as confirmed by presence of specific antibodies in the sera in ELISA assay. Western blot assays showed that the foreign gene was actually expressed in transgenic seedlings. CONCLUSIONS: The results obtained in this research provide further evidence on applicability of plant-based recombinant vaccines for protection of poultry against Newcastle disease.

Comparative proteomics and gene expression analyses revealed responsive proteins and mechanisms for salt tolerance in chickpea genotypes.

BACKGROUND: Salinity is a major abiotic stress that limits the growth, productivity, and geographical distribution of plants. A comparative proteomics and gene expression analysis was performed to better understand salinity tolerance mechanisms in chickpea. RESULTS: Ten days of NaCl treatments resulted in the differential expression of 364 reproducible spots in seedlings of two contrasting chickpea genotypes, Flip 97-43c (salt tolerant, T1) and Flip 97-196c (salt susceptible, S1). Notably, after 3 days of salinity, 80% of the identified proteins in T1 were upregulated, while only 41% in S2 had higher expression than the controls. The proteins were classified into eight functional categories, and three groups of co-expression profile. The second co-expressed group of proteins had higher and/or stable expression in T1, relative to S2, suggesting coordinated regulation and the importance of some processes involved in salinity acclimation. This group was mainly enriched in proteins associated with photosynthesis (39%; viz. chlorophyll a-b binding protein, oxygen-evolving enhancer protein, ATP synthase, RuBisCO subunits, carbonic anhydrase, and fructose-bisphosphate aldolase), stress responsiveness (21%; viz. heat shock 70 kDa protein, 20 kDa chaperonin, LEA-2 and ascorbate peroxidase), and protein synthesis and degradation (14%; viz. zinc metalloprotease FTSH 2 and elongation factor Tu). Thus, the levels and/or early and late responses in the activation of targeted proteins explained the variation in salinity tolerance between genotypes. Furthermore, T1 recorded more correlations between the targeted transcripts and their corresponding protein expression profiles than S2. CONCLUSIONS: This study provides insight into the proteomic basis of a salt-tolerance mechanism in chickpea, and offers unexpected and poorly understood molecular resources as reliable starting points for further dissection.

Comparison the Effect of Ferutinin and 17ß-Estradiol on Bone Mineralization of Developing Zebrafish (Danio rerio) Larvae.

There is an urgent need to develop novel drugs for osteoporosis which occurs due to estrogen deficiency. Phytoestrogens derived from medicinal plants would be the best alternative to chemical drugs with harmful side effects. The main purpose of the present study was to investigate the effect of ferutinin compared to 17ß-estradiol (E2) on bone mineralization of zebrafish larvae. Regarding the lack of publications, the histology analysis was performed after exposure to E2 to find effective treatment on bone mineralization of developing zebrafish larvae. Then, the larvae were exposed to four concentrations of ferutinin at three time points to assess the mortality, the expression of some related genes and histology of the ceratohyal and hyomandibular of treated larvae. The RT-PCR result of the treatment groups demonstrated the similar expression pattern in the larvae which were exposed to 1.25 µg/mL of ferutinin and 2 µM of E2 at 2 dpf, which confirmed the result of histology analysis. In addition, RT-qPCR of high concentration of ferutinin and E2 demonstrated that bmp2a/b and esr1 were downregulated and upregulated when the larvae were exposed to 5 µg/mL of ferutinin and 10 µM of E2, respectively.

Localized surface plasmon resonance biosensing of tomato yellow leaf curl virus.

Current techniques for plant virus detection, such as RT- PCR and ELISA, require multistep procedures and rely on sophisticated equipment. Due to the global spread of plant viruses, the development of simpler, faster and cheaper assay methods is inevitable. Gold nanoparticles (AuNPs) had raised much interest during recent years due to their novel optical properties or diagnostic purposes. The localized surface plasmon resonance (LSPR1) of AuNPs had been used in the development of novel colorimetric nano-biosensing systems. The frequency and intensity of the LSPR peak generally depend on the shape, size and the surrounding medium of the AuNPs. In this study, unmodified AuNPs had been used to detect the unamplified Tomato yellow leaf curl virus (TYLCV) genome in infected plants. A specific DNA probe complementary to the coat protein region of virus genome was designed. The extracted total DNA of uninfected and infected plants was mixed with hybridization buffer and the designed probe. The mixture was denatured, annealed and then cooled to room temperature and was followed by AuNPs addition. The color changes in the samples indicating the presence of target virus infections were assessed visually after the addition of salt and confirmed by UV-Vis spectroscopy. The results showed that this strategy allowed for fast and sensitive detection of TYLCV genome and eliminated the need for PCR amplification and detection equipment.

A Study to Assess the Role of Gluten Encoded Genes and Their Regulatory Elements in Bread Making Quality of Wheat.

BACKGROUND: Quality of bread baking is affected by gluten genes and balance between their expressions. Hence, it is necessary for a comprehensive research to study and compare all gluten genes and their regulating elements simultaneously. OBJECTIVES: The aim of this study was to evaluate the molecular mechanism of bread quality at the level of coding genes and regulating elements via comparative transcriptome analysis of two extreme wheat cultivars. MATERIALS AND METHODS: RNAs were extracted from the grain of two wheat cultivars with high (Pishtaz) and low (Navid) bread making qualities, collected during endosperm development at five stages. mRNAs were sequenced and gluten transcripts were assessed to find differentially expressed genes. Then, transcription factors interacting with gluten genes were detected and evaluated for expression. RESULTS: Results showed that Ɣ-gliadin and LMW-GS genes had a higher expression in Pishtaz and Navid, respectively. Most identified transcription factors were active at the early stage of growth and it seemed that NAC and ERF transcription factors had significant roles in regulating genes with different expressions. There was no significant difference in the expression level of NACs between two cultivars. It is proposed that the ERF transcription factor which classified as BREB2C transcription factor could control the expression of LMW-GS genes in two cultivars and functionally act as a repressor for their target genes. CONCLUSION: The priority of Pishtaz wheat cultivar in bread quality originated from high expression levels of Ɣ-gliadin gene and ERF transcription factor.

Transient expression of CCL21as recombinant protein in tomato.

The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum, the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

Inhibition of quorum sensing in Pseudomonas aeruginosa by two herbal essential oils from Apiaceae family.

Ferula (Ferula asafoetida L.) and Dorema (Dorema aucheri Bioss.) both from Apiaceae family were tested for their anti-quorum sensing (QS) activity against Pseudomonas aeruginosa. Both essential oils exhibited anti-QS activity at 25 µg/ml of concenteration. At this concenteration Ferula fully abolished and Dorema reduced the violacein production by C. violaceum. Pyocyanin, pyoverdine, elastase and biofilm production were decreased in Ferula oil treatments. Dorema oil reduced pyoverdine and elastase production, while pyocyanin and biofilm production were not affacted. Expresion analysis of QS-dependent genes confirmed our phenotypic data. Our data introduced native Dorema and Ferula plants as novel QS and virulence inhibitors.

Genetic manipulation, a feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production.

Developing a reliable technique to transform unicellular green algae, Chlorella vulgaris, could boost potentials of using microalgae feedstock in variety of applications such as biodiesel production. Volumetric lipid productivity (VLP) is a suitable variable for evaluating potential of algal species. In the present study, the highest VLP level was recorded for C. vulgaris (79.08 mg l(-1 )day(-1)) followed by 3 other strains studied; C. emersonii, C. protothecoides, and C. salina by 54.41, 45 and 18.22 mg l(-1)day(-1), respectively. Having considered the high productivity of C. vulgaris, it was selected for the preliminary transformation experiment through micro-particle bombardment. Plasmid pBI 121, bearing the reporter gene under the control of CaMV 35S promoter and the kanamycin marker gene, was used in cells bombardment. Primary selection was done on a medium supplemented by 50 mg l(-1) kanamycin. After several passages, the survived cells were PCR-tested to confirm the stability of transformation and then were found to exhibit ß-glucuronidase (GUS) activity in comparison with the control cells. Southern hybridization of npt II probe with genomic DNA revealed stable integration of the cassette in three different positions in the genome. The whole process was successfully implemented as a pre-step to transform the algal cells by genes involved in lipid production pathway which will be carried out in our future studies.

Optimizing regeneration condition in chickpea (Cicer arietinum L.).

In this study, multiple shoot induction and whole plant regeneration from decapitated embryo axes of three chick peal genotypes including MCC252, MCC283 and MCC505 were evaluated on modified Murashige and Skoog's medium (MMS) which, its vitamins were replaced by vitamins of B5 medium, supplemented with varied concentration of thidiazuron (0.1, 0.2 mg L(-1)) or 6-benzylaminopurin (1,2 mg L(-1)) or zeatin (1, 2 mg L(-1)) treatments. BAP was found to be the most effective cytokinin in normal multiple shoot induction. Shoots were elongated on growth regulator-free medium and then rooted on two media containing 1/4 MMS salts and B5 vitamins + 3% sucrose + 0.8% agar with indol-3-butyric acid (0.4 or 1 mg L(-1)). The highest rooting frequency resulted in a medium including 0.4 mg L(-1) IBA. It was found that different shoot induction media also positively affected rooting, where a medium with 2 mg L(-1) BAP in MCC252/MCC505 and a medium with 2 mg L(-1) zeatin in MCC283 were the best media in shoot induction that produced high frequency, thick spread roots. Plantlets were preliminary acclimatized in liquid medium (1/4 MMS salts and B5 vitamins + 3% sucrose + 0.4 mg L(-1) IBA) for 7 to 14 days, then transferred to pots filled by cocopit: perlite (1:1) and kept in a growth chamber until their shoots and roots were well developed. This resulted in more than 70% survival rate.