Comparative Genomics, Pangenome, and Phylogenomic Analyses of Brenneria spp., and Delineation of Brenneria izadpanahii sp. nov.

Brenneria species are bacterial plant pathogens mainly affecting woody plants. Association of all members with devastating disorders (e.g., acute oak decline in Iran and United Kingdom) are due to adaptation and pathogenic behavior in response to host and environmental factors. Some species, including B. goodwinii, B. salicis, and B. nigrifluens, also show endophytic residence. Here we show that all species including novel Brenneria sp. are closely related. Gene-based and genome/pangenome-based phylogeny divide the genus into two distinct lineages, Brenneria clades A and B. The two clades were functionally distinct and were consistent with their common and special potential activities as determined via annotation of functional domains. Pangenome analysis demonstrated that the core pathogenicity factors were highly conserved, an hrp gene cluster encoding a type III secretion system was found in all species except B. corticis. An extensive repertoire of candidate virulence factors was identified. Comparative genomics indicated a repertoire of plant cell wall degrading enzymes, metabolites/antibiotics, and numerous prophages providing new insights into Brenneria-host interactions and appropriate targets for further characterization. This work not only documented the genetic differentiation of Brenneria species but also delineates a more functionally driven understanding of Brenneria by comparison with relevant Pectobacteriaceae thereby substantially enriching the extent of information available for functional genomic investigations.

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein.

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the αB-αB" domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

Distribution and Variation of Bacterial Endosymbiont and "Candidatus Liberibacter asiaticus" Titer in the Huanglongbing Insect Vector, Diaphorina citri Kuwayama.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.

Attenuation and quantitation of virulence gene expression in quorum-quenched Dickeya chrysanthemi.

N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18 % in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

Phylogenetic relationships, recombination analysis, and genetic variability among diverse variants of tomato yellow leaf curl virus in Iran and the Arabian Peninsula: further support for a TYLCV center of diversity.

The discovery of five strains of TYLCV in Iran, including the most well-known and widespread, TYLCV-IL, spurred a detailed study of the full-length genomes of additional TYLCV field isolates and an in-depth analysis of phylogenetic relationships, extent of recombination, and genetic variability of TYLCV isolates within Iran and throughout the Arabian Peninsula. Phylogenetic analysis of complete genome sequences of TYLCV isolates from Iran and other countries revealed four monophyletic clusters could be differentiated based on geographical origin, indicating that recent dispersal of these populations (by the vector or by humans) from these four regions has occurred minimally, or not at all. Genetic analysis revealed that TYLCV-IL isolates from southern Iran possessed greater genetic variability than the northeastern isolates, a pattern that may be reflective of evolution driven by geographically dependent isolation. Similarly, isolates of TYLCV-OM originating from Oman showed greater genetic variability than TYLCV-OM variants from Iran. Major recombination events, which were detected in all strains of TYLCV had breakpoints initiating in the C1, C1/C4, C2/C3 and V1 open reading frames (ORFs) and ending at the non-coding region and the C1, C1/C2 and C3 ORFs. Hence, these regions have consistently served as hot spots for recombination worldwide during the evolution of all currently recognized isolates and strains of TYLCV.

Identification of the chemical profile and evaluation of the antimicrobial effect of Eryngium billardieri Delar essential oil component against bacterial species of agricultural and food interest.

Studies on the antibacterial activity of the essential oil of E. billardieri are limited. In this study, we identified this herb as a natural complex effective against several bacteria by employing disk diffusion and broth microdilution susceptibility methods. Primary estimation of the antimicrobial effect of this herbal compound by disk diffusion method showed that the oil could inhibit the growth of the tested bacteria by the appearance of haloes between 8.25 and 21.25 mm. In the next step, the oil was found to be active against all 24 tested Gram-negative and Gram-positive bacteria in the broth media, at minimum inhibitory concentrations ranging from 0.67 to 34.17 g L-1. Furthermore, Enterococcus faecalis and Curtobacterium flaccumfaciens pv. flaccumfaciens were the most sensitive food and plant pathogenic bacteria, respectively. Gas chromatography-mass spectrometry analysis was conducted to assign the ingredients present in the oil; 34 different components representing 95.71% of the total oil were identified, with n-hexadecanoic acid being the dominant component, followed by 2-Pentadecanone, 6,10,14-trimethyl, 1H-Indene, 1-ethylideneoctahydro-, and Cinnamyl tiglate. These findings demonstrate, for the first time, a broad spectrum of the antibacterial capacity of E. billardieri. Based on these observations, the oil could be applied as a natural preservative with the potential for designing novel products. Its bioactive agents can also be isolated for further use in the food and agricultural industries.

Critical points for the design and application of RNA silencing constructs for plant virus resistance.

Control of plant virus diseases is a big challenge in agriculture as is resistance in plant lines to infection by viruses. Recent progress using advanced technologies has provided fast and durable alternatives. One of the most promising techniques against plant viruses that is cost-effective and environmentally safe is RNA silencing or RNA interference (RNAi), a technology that could be used alone or along with other control methods. To achieve the goals of fast and durable resistance, the expressed and target RNAs have been examined in many studies, with regard to the variability in silencing efficiency, which is regulated by various factors such as target sequences, target accessibility, RNA secondary structures, sequence variation in matching positions, and other intrinsic characteristics of various small RNAs. Developing a comprehensive and applicable toolbox for the prediction and construction of RNAi helps researchers to achieve the acceptable performance level of silencing elements. Although the attainment of complete prediction of RNAi robustness is not possible, as it also depends on the cellular genetic background and the nature of the target sequences, some important critical points have been discerned. Thus, the efficiency and robustness of RNA silencing against viruses can be improved by considering the various parameters of the target sequence and the construct design. In this review, we provide a comprehensive treatise regarding past, present and future prospective developments toward designing and applying RNAi constructs for resistance to plant viruses.

Genetic analysis of Iranian population of Potato leafroll virus based on ORF0.

Potato leafroll virus (PLRV) is a destructive virus of potatoes and responsible for high yield losses wherever potatoes are grown. In this study, DNA fragments containing ORF0 from each of nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates from 12 different countries including 14 Iranian isolates showed that the identities of ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96-100 % and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of full-length ORF0 and overlapping and non-overlapping regions of ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identified negative selection with different ratios for each of the mentioned genomic regions suggesting effects of F-box motif and -1 frameshift on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role for this phenomenon in influencing genetic diversity within this virus population.

Neoscytalidium novaehollandiae causes dieback on Pinus eldarica and its potential for infection of urban forest trees.

Neoscytalidium novaehollandiae is one of the most important pathogens on woody plants which has increasingly been reported as a pathogen in different hosts in recent years. The pine trees are widely cultured in many cities of Iran. In recent years, dieback symptoms were observed on Pinus eldarica trees in Tehran and Qazvin provinces. The aim of this study was to investigate the dieback causal agent on P. eldarica trees in Iran. The branches and cones of P. eldarica trees were sampled for fungal isolation. The morphological and molecular characterizations (ITS, LSU, and TEF1-α regions) identified N. novaehollandiae as a dieback causal agent. This is the first report of N. novaehollandiae disease of P. eldarica trees in Iran. Furthermore, disease severity was assayed on 19 urban forest trees under three different temperature and relative humidity (RHs) regimes. C regime (29 °C and 15% RH) displayed more disease severity on detached branches than B (24 °C and 80% RH) and A (19 °C and 35% RH) ones. This study presents the host range of this pathogen, and showed that these potential hosts are prone to this pathogen under high temperature and low humidity which urban forest trees experienced in recent decades.

Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Identification of a defense response gene involved in signaling pathways against PVA and PVY in potato.

Potato is the most important non-grain food crop in the world. Viruses, particularly potato virus Y (PVY) and potato virus A (PVA), are among the major agricultural pathogens causing severe reduction in potato yield and quality worldwide. Virus infection induces host factors to interfere with its infection cycle. Evaluation of these factors facilitates the development of intrinsic resistance to plant viruses. In this study, a small G-protein as one of the critical signaling factors was evaluated in plant response to PVY and PVA to enhance resistance. For this purpose, the gene expression dataset of G-proteins in potato plant under five biotic (viruses, bacteria, fungi, nematodes, and insects) and four abiotic (cold, heat, salinity, and drought) stress conditions were collected from gene expression databases. We reduced the number of the selected G-proteins to a single protein, StSAR1A, which is possibly involved in virus inhibition. StSAR1A overexpressed transgenic plants were created via the Agrobacterium-mediated method. Real-time PCR and Enzyme-linked immunosorbent assay tests of transgenic plants mechanically inoculated with PVY and PVA indicated that the overexpression of StSAR1A gene enhanced resistance to both viruses. The virus-infected transgenic plants exhibited a greater stem length, a larger leaf size, a higher fresh/dry weight, and a greater node number than those of the wild-type plants. The maximal photochemical efficiency of photosystem II, stomatal conductivity, and net photosynthetic rate in the virus-infected transgenic plants were also obviously higher than those of the control. The present study may help to understand aspects of resistance against viruses.

Efficient silencing gene construct for resistance to multiple common bean (Phaseolus vulgaris L.) viruses.

One promising strategy to engineer plants that are resistant to plant pathogens involves transforming plants with RNA silencing constructs for resistance to multiple pathogens. Garden bean is significantly damaged by bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV) and cucumber mosaic virus (CMV). In this study, we prepared constructs producing sense, antisense and hairpin RNA (hpRNA) structures to target single as well as multiple viruses. Silencing efficiency of these constructions was analyzed using Agrobacterium (GV3101) transient expression in Nicothinia bethamiana and Phaseolus vulgaris plants. The results showed significantly reduced disease symptoms and virus accumulation in N. bethamiana plants. Generally, the efficiency of the prepared constructs was hairpin, antisense and sense, respectively, and also, there was a significant difference between mono-gene and multiple-gene constructs for reducng virus accumulation and the multiple-gene constructs showed higher effectiveness. Experiments in this study showed that using Agrobacterium harboring binary constructs containing a Caenorhabditis elegans gene, Ced-9, or a plant gene, AtBag-4, anti-apoptosis gene as a mix suspension with an Agrobacterium containing pFGC-BNC.h, a plasmid containing multiple gene fragments consisting of BCMV-CP, BCMNV-HC-Pro and CMV-2b, improved the efficiency of pFGC-BNC.h transformation. We showed reduced virus accumulation in these transgenic bean plans.

The Role of Cell Wall Degrading Enzymes in Antagonistic Traits of Trichoderma virens Against Rhizoctonia solani.

BACKGROUND: High antagonistic ability of different Trichoderma species against a diverse range of plant pathogenic fungi has led them to be used as a biological fungicide in agriculture. They can also promote plant growth, fertility, resistance to stress, and absorption of nutrients. They are also opportunistic and symbiotic pathogens, which can lead to the activation of plant defense mechanisms. OBJECTIVES: The aim of this present study was to investigate possible enhancement of lytic enzymes production and biocontrol activity of T. virens against Rhizoctonia solani through gamma radiation and to find the relationship between changes in lytic enzyme production and antagonistic activity of T. virens. MATERIAL AND METHODS: Dual culture conditions were used to evaluate the antagonistic effect of T. virens and its gamma mutants against R. solani. Then, their chitinase and cellulase activities were measured. For more detailed investigation of enzymes, densitometry pattern of the proteins was extracted from the T. virens wild-type and its mutants were obtained via SDS-polyacrylamide gel electrophoresis. RESULTS: The mutant T.vi M8, T. virens wild-type and mutant T.vi M20 strains showed the maximum antagonistic effects against the pathogen, respectively. Data showed that the mutant T. vi M8 reduced the growth of R. solani by 58 %. The mutants revealed significantly different (p<0.05) protein contents, chitinase and cellulase production (mg.mL-1) and activity (U.mL-1) compared to the wild-type strain. The highest extracellular protein production in the supernatant of chitinase and cellulase TFM was observed for the T.vi M11 and T.vi M17 strains, respectively. The T.vi M12 and wild-type strains secreted chitinase and cellulase significantly more than other strains did. Densitometry of SDS-PAGE gel bands indicated that both the amount and diversity of chitinase related proteins in the selected mutant (T. vi M8) were far higher than those of the wild-type. The diversity of molecular weight of proteins extracted from the T. virens M8 (20 proteins or bands) was very high compared to the wild-type (10 proteins) and mutant T.vi M15 (2 proteins). CONCLUSIONS: Overall, there was a strong link between the diversity of various chitinase proteins and the antagonistic properties of the mutant M8.

Color morphology of Diaphorina citri influences interactions with its bacterial endosymbionts and 'Candidatus Liberibacter asiaticus'.

Diaphorina citri is a vector of 'Candidatus Liberibacter asiaticus,' (CLas), associated with Huanglongbing, (HLB, or citrus greening) disease in citrus. D. citri exhibits three different color morph variants, blue, gray and yellow. Blue morphs have a greater capacity for long-distance flight as compared to non-blue morphs, but little else is known about how color morphology influences vector characteristics. In this study, we show that the color morphology of the insect is derived from pigmented cells of the fat body. Blue morphs acquire a lower level of CLas in their bodies from infected trees as compared to their gray and yellow conspecifics, referred to in this paper collectively as non-blue morphs. Accordingly, CLas titer in citrus leaves inoculated by non-blue insects was 6-fold higher than in leaves inoculated by blue insects. Blue color morphs harbored lower titers of Wolbachia and 'Candidatus Profftella armatura,' two of the D. citri bacterial endosymbionts. Expression of hemocyanin, a copper-binding oxygen transport protein responsible for the blue coloration of hemolymph of other arthropods and mollusks, was previously correlated with blue color morphology and is highly up-regulated in insects continuously reared on CLas infected citrus trees. Based on our results, we hypothesized that a reduction of hemocyanin expression would reduce the D. citri immune response and an increase in the titer of CLas would be observed. Surprisingly, a specific 3-fold reduction of hemocyanin-1 transcript levels using RNA silencing in blue adult D. citri morphs had an approximately 2-fold reduction on the titer of CLas. These results suggest that hemocyanin signaling from the fat body may have multiple functions in the regulation of bacterial titers in D. citri, and that hemocyanin is one of multiple psyllid genes involved in regulating CLas titer.

The Effect of Methyl Jasmonate and Temperature on the Transient Expression of Recombinant Proteins in Cucurbita pepo L.

The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference between applied treatments. The optimized treatment significantly increased the expression of rtPA compared to the control. The Real-Time PCR reaction for both GFP and rtPA genes showed no significant differences between optimum and control treatments, however, transcripts of the small subunit of RuBisCO were extremely down-regulated in optimum treatment condition. Reduction in RuBisCO expression at protein level was tangible under treatment condition based on the ELISA test. Therefore, it can be inferred that suppressing the expression of RuBisCO, probably resulted in higher access of expression system to free amino acids inside the cell. In this study, MeJA has been shown to be a positive factor, but the low temperature (17 °C), unlike previous studies, suppressed the expression of recombinant protein unexpectedly, probably due to the incompatibility of the viral construct with low temperature. In conclusion, the use of a suitable gene construct, which is not sensitive to temperature, is likely to result in a more favorable outcome.

Identification and expression analysis of a microRNA cluster derived from pre-ribosomal RNA in Papaver somniferum L. and Papaver bracteatum L.

Opium poppy (Papaver somniferum L.) is one of the ancient medical crops, which produces several important alkaloids such as morphine, noscapine, sanguinarine and codeine. MicroRNAs are endogenous non-coding RNAs that play important regulatory roles in plant diverse biological processes. Many plant miRNAs are encoded as single transcriptional units, in contrast to animal miRNAs, which are often clustered. Herein, using computational approaches, a total of 22 miRNA precursors were identified, which five of them were located as a clustered in pre-ribosomal RNA. Afterward, the transcript level of the precursor and the mature of clustered miRNAs in two species of the Papaveraceae family, i.e. P. somniferum L. and P. bracteatum L, were quantified by RT-PCR. With respect to obtained results, these clustered miRNAs were expressed differentially in different tissues of these species. Moreover, using target prediction and Gene Ontology (GO)-based on functional classification indicated that these miRNAs might play crucial roles in various biological processes as well as metabolic pathways. In this study, we discovered the clustered miRNA derived from pre-rRNA, which may shed some light on the importance of miRNAs in the plant kingdom.

Shannon Entropy to Evaluate Substitution Rate Variation Among Viral Nucleotide Positions in Datasets of Viral siRNAs.

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.

Transient expression of anti-VEFGR2 nanobody in Nicotiana tabacum and N. benthamiana.

In human, the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) is critical for tumor angiogenesis. This is a vital process for cancer tumor growth and metastasis. Blocking VEGF/VEGFR2 conjugation by antibodies inhibits the neovascularization and tumor metastasis. This investigation designed to use a transient expression platform for production of recombinant anti-VEGFR2 nanobody in tobacco plants. At first, anti-VEGFR2-specific nanobody gene was cloned in a Turnip mosaic virus (TuMV)-based vector, and then, it was expressed in Nicotiana benthamiana and Nicotiana tabacum cv. Xanthi transiently. The expression of nanobody in tobacco plants were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), dot blot, enzyme-linked immunosorbent assays (ELISA), and Western blot analysis. It was shown that tobacco plants could accumulate nanobody up to level 0.45% of total soluble protein (8.3 µg/100 mg of fresh leaf). This is the first report of the successful expression of the camelied anti-VEFGR2 nanobody gene in tobacco plants using a plant viral vector. This system provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in tobacco plants.

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing.

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 degrees C, but not at 33 degrees C. By contrast, systemic infection at 33 degrees C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 degrees C, but not at 33 degrees C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.

Bacillus subtilis affects miRNAs and flavanoids production in Agrobacterium-Tobacco interaction.

Agrobacterium tumefaciens is a very destructive plant pathogen. Selection of effective biological agents against this pathogen depends on more insight into molecular plant defence responses during the biocontrol agent-pathogen interaction. Auxin as a phytohormone is a key contributor in pathogenesis and plant defence and accumulation of auxin transport carriers are accompanied by increasing in flavonoid and miRNAs concentrations during plant interactions with bacteria. The aim of this research was molecular analysis of Bacillus subtilis (ATCC21332) biocontrol effect against A. tumefaciens (IBRC-M10701) pathogen interacting with Nicotiana tabacum plants. Tobacco plants were either treated with both or one of the challenging bacteria and the expression of miRNAs inside the plants were analysed through qRT-PCR. The results indicated that the bacterial treatments affect expression level of nta-miRNAs. In tobacco plants treated only with A. tumefaciens the expression of nta-miR393 was more than that was recorded for nta-miR167 (3.8 folds, P < 0.05 in 3dpi). While the expression level of nta-miR167 was more than the expression of nta-miR393 in other treatments including tobacco plants treated only with B. subtilis (2.1 folds, P < 0.05) and the plants treated with both of the bacteria (3.9 folds, P < 0.05) in 3 dpi. Also, the composition and concentration of rutin, myrecetin, daidzein and vitexin flavanoid derivatives were detected using HPLC and analysed according the standard curves. All of the tested flavanoid compounds were highly detected in Tobacco plants which were only challenged with A. tumefaciens. The amount of these compounds in the plants which were challenged with the B. subtilis alone, was similar to the amount recorded for the plants challenged with the both bacteria. This study suggests a relationship between the upregulation of nta-miR167, nta-miR393 and accumulation of flavanoid compounds. Overall, the expression of these miRNAs as well as flavonoid derivatives has the potential of being used as biomarkers for the interaction of B. subtilis and A. tumefaciens model system in N. tabacum.