Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.

BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.

Antibacterial Activity of Endophytic Bacteria Against Sugar Beet Root Rot Agent by Volatile Organic Compound Production and Induction of Systemic Resistance.

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by the endophytic bacteria Streptomyces sp. B86, Pantoea sp. Dez632, Pseudomonas sp. Bt851, and Stenotrophomonas sp. Sh622 isolated from healthy sugar beet (Beta vulgaris) and sea beet (Beta maritima) were evaluated for their effects on the virulence traits of Bacillus pumilus Isf19, the causal agent of harvested sugar beet root rot disease. The gas chromatographymass spectrometry (GC-MS) analysis revealed that B86, Dez632, Bt851, and Sh622 produced 15, 28, 30, and 20 VOCs, respectively, with high quality. All antagonistic endophytic bacteria produced VOCs that significantly reduced soft root symptoms and inhibited the growth of B. pumilus Isf19 at different levels. The VOCs produced by endophytic bacteria significantly reduced swarming, swimming, and twitching motility by B. pumilus Isf19, which are important to pathogenicity. Our results revealed that VOCs produced by Sh622 and Bt851 significantly reduced attachment of B. pumilus Isf19 cells to sugar beetroots, and also all endophytic bacteria tested significantly reduced chemotaxis motility of the pathogen toward root extract. The VOCs produced by Dez632 and Bt851 significantly upregulated the expression levels of defense genes related to soft rot resistance. Induction of PR1 and NBS-LRR2 genes in sugar beetroot slices suggests the involvement of SA and JA pathways, respectively, in the induction of resistance against pathogen attack. Based on our results, the antibacterial VOCs produced by endophytic bacteria investigated in this study can reduce soft rot incidence.

Hairy root induction and Farnesiferol B production of endemic medicinal plant Ferula pseudalliacea.

The effects of medium, gibberellic acid (GA3) and stratification treatments on the seed germination of Ferula pseudalliacea were evaluated. Filter paper medium, 500 micro molar GA3 and 8 week chilling treatment were resulted in significantly more seed germination than others. F. pseudalliacea was also transformed by Agrobacterium rhizogenes. Explants from young leaves, stems, cotyledon, and embryo were inoculated with A. rhizogenes strains ATCC 15834, 1724, A4, LB9402 and Ar318. Hairy roots were induced only from 10 to 12-days embryo explants using strains ATCC 15824 and 1724. Although, the transformation efficiency of ATCC 15834 (4%) strain was higher than 1724 (2%). Maximum hairy root transformation frequency (25%) was obtained in infection time of 10 min compared to that of 20 (20%) and 30 (5%) min. In addition, the transformation rate was significantly higher at the inoculation time of 72 h (29%) compared to that of 48 h (22%) and 24 h (6%). Transgenic hairy root lines were confirmed by PCR amplification of rolB gene. Hairy root lines were produced higher biomass in half B5 medium compared to that of half MS medium. Hairy roots lines from the strain ATCC 15834 produced more hairy root numbers and fresh and dried biomass compared to that of the strain 1724. Analyses of transgenic hairy root and natural roots extracts using HPLC showed that all the hairy root lines produced farnesiferol B.

Antifungal activity of volatile compounds produced by Staphylococcus sciuri strain MarR44 and its potential for the biocontrol of Colletotrichum nymphaeae, causal agent strawberry anthracnose.

A nonpathogenic endophytic bacterial isolate, recovered from Fragaria × ananassa stolon, and its antifungal activity against Colletotrichum nymphaeae was evaluated under in vitro, in vivo, and greenhouse conditions. Bacterial isolate was identified as Staphylococcus sciuri MarR44 (Strain ID: WDCM 891 = CCSM-B 00640) using phenotypic and biochemical properties and molecular phylogenetic analysis of the 16S rDNA gene sequences. The living cells of strain MarR44 inhibited mycelial growth of C. nymphaeae (52.46%) using dual-culture method. The volatile compounds (VOCs) produced by MarR44 inhibited mycelial growth and conidial germination of C. nymphaeae by 34.52% and 82.81%, respectively. However, inhibition percentage of mycelial growth of pathogen by culture filtrates of the strain MarR44 was lower (23.07%) than that for the two dual culture and volatile compounds assay tests. Moreover, the cell-free-culture filtrates of this strain reduced the biomass and conidial germination of pathogen by 91.89% and 41.10%, respectively. Also, the strain MarR44 was capable of producing protease, chitinase, HCN, siderophore, IAA, gibberellin, and biofilm. The living cells and volatile compounds of the strain MarR44 reduced anthracnose disease at post-harvest on fruit by 52.45% and 72.17%, respectively. Furthermore, disease severity of strawberry anthracnose was reduced using drenching soil and inoculated plants methods by 77.77 and 72.22%, respectively, 60 days after inoculation. The VOCs released by strain MarR44 were analyzed by Gas chromatography-mass spectroscopy (GC-MS). Out of 24 identified VOCs, Mesityl oxide (81.436%), Acetic acid, 2-methylpropyl ester (3.442%), 4-Methyldecane (1.837%), 4-Penten-2-one,4-methyl- (1.739%), Toluene (1.248%), and o-Xylene (1.24%) were the major components. The mode of action of S. sciuri MarR44 on the C. nymphaeae was through the production of antifungal volatile compounds (Antibiosis), which inhibited mycelial growth and conidial germination of pathogen in vitro and fruit decay development in vivo. To the best of our knowledge, this is the first report of S. sciuri having antifungal activity against causal agent strawberry anthracnose. These results indicated that the VOCs of S. sciuri strain MarR44 are promising biofumigant for management of strawberry anthracnose.

A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation.

Agrobacterium rhizogenes, along with A. tumefaciens, has been used to affect genetic transformation in plants for many years. Detailed studies conducted in the past have uncovered the basic mechanism of foreign gene transfer and the implication of Ri/Ti plasmids in this process. A number of reviews exist describing the usage of binary vectors with A. tumefaciens, but no comprehensive account of the numerous binary vectors employed with A. rhizogenes and their successful applications has been published till date. In this review, we recollect a brief history of development of Ri-plasmid/Ri-T-DNA based binary vectors systems and their successful implementation with A. rhizogenes for different applications. The modification of native Ri plasmid to introduce foreign genes followed by development of binary vector using Ri plasmid and how it facilitated rapid and feasible genetic manipulation, earlier impossible with native Ri plasmid, have been discussed. An important milestone was the development of inducible plant expressing promoter systems which made expression of toxic genes in plant systems possible. The successful application of binary vectors in conjunction with A. rhizogenes in gene silencing and genome editing studies which are relatively newer developments, demonstrating the amenability and adaptability of hairy roots systems to make possible studying previously intractable research areas have been summarized in the present review.

Hairy root culture optimization and resveratrol production from Vitis vinifera subsp. sylvesteris.

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.

In silico identification, phylogenetic and bioinformatic analysis of argonaute genes in plants.

Argonaute protein family is the key players in pathways of gene silencing and small regulatory RNAs in different organisms. Argonaute proteins can bind small noncoding RNAs and control protein synthesis, affect messenger RNA stability, and even participate in the production of new forms of small RNAs. The aim of this study was to characterize and perform bioinformatic analysis of Argonaute proteins in 32 plant species that their genome was sequenced. A total of 437 Argonaute genes were identified and were analyzed based on lengths, gene structure, and protein structure. Results showed that Argonaute proteins were highly conserved across plant kingdom. Phylogenic analysis divided plant Argonautes into three classes. Argonaute proteins have three conserved domains PAZ, MID and PIWI. In addition to three conserved domains namely, PAZ, MID, and PIWI, we identified few more domains in AGO of some plant species. Expression profile analysis of Argonaute proteins showed that expression of these genes varies in most of tissues, which means that these proteins are involved in regulation of most pathways of the plant system. Numbers of alternative transcripts of Argonaute genes were highly variable among the plants. A thorough analysis of large number of putative Argonaute genes revealed several interesting aspects associated with this protein and brought novel information with promising usefulness for both basic and biotechnological applications.

Identification and characterization of a NBS-LRR class resistance gene analog in Pistacia atlantica subsp. Kurdica.

P. atlantica subsp. Kurdica, with the local name of Baneh, is a wild medicinal plant which grows in Kurdistan, Iran. The identification of resistance gene analogs holds great promise for the development of resistant cultivars. A PCR approach with degenerate primers designed according to conserved NBS-LRR (nucleotide binding site-leucine rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from P. atlantica subsp. Kurdica. A DNA fragment of the expected 500-bp size was amplified. The nucleotide sequence of this amplicon was obtained through sequencing and the predicted amino acid sequence compared to the amino acid sequences of known R-genes revealed significant sequence similarity. Alignment of the deduced amino acid sequence of P. atlantica subsp. Kurdica resistance gene analog (RGA) showed strong identity, ranging from 68% to 77%, to the non-toll interleukin receptor (non-TIR) R-gene subfamily from other plants. A P-loop motif (GMMGGEGKTT), a conserved and hydrophobic motif GLPLAL, a kinase-2a motif (LLVLDDV), when replaced by IAVFDDI in PAKRGA1 and a kinase-3a (FGPGSRIII) were presented in all RGA. A phylogenetic tree, based on the deduced amino-acid sequences of PAKRGA1 and RGAs from different species indicated that they were separated in two clusters, PAKRGA1 being on cluster II. The isolated NBS analogs can be eventually used as guidelines to isolate numerous R-genes in Pistachio.

Differential expression of eight defensin genes of N. benthamiana following biotic stress, wounding, ethylene, and benzothiadiazole treatments.

Eight Nicotiana benthamiana defensin genes were identified that could be divided into two classes with class II defensins being longer than class I defensins due to an additional acidic C-terminal domain. Class I defensins were NbDef1.1, NbDef1.2, NbDef1.3, NbDef1.4, NbDef1.5, and NbDef1.6, and class II were Nbdef2.1 and NbDef2.2. Relative RT-PCR showed that NbDef1.1, NbDef1.2, and NbDef1.4 had relatively similar expression levels in healthy leaves, stems, roots, flowers, and seeds. However, Nbdef1.3, NbDef1.5, and NbDef1.6 had varying degrees of tissue specific expression, and Nbdef2.1 and NbDef2.2 had strictly flower-specific expression. None of the defensins were significantly induced by infection by Colletotrichum destructivum or C. orbiculare. However, infection by Pseudomonas syringae pv. tabaci resulted in increased expression of Nbdef1.2 and Nbdef2.2, and decreased expression of NbDef1.1, NbDef1.4, and NbDef1.6. In the hypersensitive response of N. benthamiana containing Pto with P. syringae pv. tabaci containing AvrPto, only NbDef2.2 was significantly up-regulated. Expression of the genes was also affected by abiotic treatments. Both wounding and ethylene treatments resulted in a strong induction of NbDef2.2 and a moderate to weak induction of NbDef1.1, NbDef1.2, and NbDef1.4. Only weak or no induction was observed with treatment with benzothiadiazole. The expression of these eight defensin genes demonstrates that only a small fraction of the members of a defensin gene family will respond to a particular hemibiotrophic pathogen as well as to abiotic stress or signaling molecules.