Ali Taheri

Prunus domestica Pathogenesis-Related Protein-5 Activates the Defense Response Pathway and Enhances the Resistance to Fungal Infection

Pathogenesis-related protein-5 (PR-5) has been implicated in plant disease resistance and its antifungal activity has been demonstrated in some fruit species. However, their roles, especially their interactions with the other defense responses in plant cells, are still not fully understood. In this study, we have cloned and characterized a new PR-5 cDNA named PdPR5-1 from the European plum (Prunus domestica). Expression of PdPR5-1 was studied in different cultivars varying in resistance to the brown rot disease caused by the necrotrophic fungus Monilinia fructicola. In addition transgenic Arabidopsis, ectopically expressing PdPR5-1 was used to study its role in other plant defense responses after fungal infection. We show that the resistant cultivars exhibited much higher levels of transcripts than the susceptible cultivars during fruit ripening. However, significant rise in the transcript levels after infection with M. fructicola was observed in the susceptible cultivars too. Transgenic Arabidopsis plants exhibited more resistance to Alternaria brassicicola. Further, there was a significant increase in the transcripts of genes involved in the phenylpropanoid biosynthesis pathway such as phenylalanine ammonia-lyase (PAL) and phytoalexin (camalexin) pathway leading to an increase in camalexin content after fungal infection. Our results show that PdPR5-1 gene, in addition to its anti-fungal properties, has a possible role in activating other defense pathways, including phytoalexin production.

Expression analysis of a plum pathogenesis related 10 (PR10) protein during brown rot infection

Plant PR10 is one of the pathogenesis related proteins, induced upon exposure to different stress conditions including fungal infection. PR10 proteins have been implicated in fungal disease resistance in some species; however its transcriptional regulation is not well understood. In the present work we cloned a PR10 gene from European plums (Prunus domestica L.) and monitored the quantitative changes in its transcript levels as a result of fungal infection in two varieties. We also studied the possible involvement of the membrane degrading enzyme phospholipase D-alpha (PLDα). In the susceptible variety, ‘Veeblue’, infection with the brown rot fungus Monilinia fructicola induced PLDα and PR10 expression, while in the resistant variety, ‘Violette’, a constitutive expression of PLDα and PR10 transcripts levels were observed. Resistance to M. fructicola also coincides with a sharp decrease in the expression of ABI1, a protein phosphatase and elevated hydrogen peroxide content after infection. Further, inhibition of PLDα by hexanal treatment, up-regulated ABI1 and decreased PR10 expression, suggesting a possible relationship between the two. We further confirm these results in Arabidopsis abi1 mutant that shows a higher level of PR10 transcripts.

The compact genome of the plant pathogen Plasmodiophora brassicae is adapted to intracellular interactions with host Brassica spp

BackgroundThe protist Plasmodiophora brassicae is a soil-borne pathogen of cruciferous species and the causal agent of clubroot disease of Brassicas including agriculturally important crops such as canola/rapeseed (Brassica napus). P. brassicae has remained an enigmatic plant pathogen and is a rare example of an obligate biotroph that resides entirely inside the host plant cell. The pathogen is the cause of severe yield losses and can render infested fields unsuitable for Brassica crop growth due to the persistence of resting spores in the soil for up to 20xa0years.ResultsTo provide insight into the biology of the pathogen and its interaction with its primary host B. napus, we produced a draft genome of P. brassicae pathotypes 3 and 6 (Pb3 and Pb6) that differ in their host range. Pb3 is highly virulent on B. napus (but also infects other Brassica species) while Pb6 infects only vegetable Brassica crops. Both the Pb3 and Pb6 genomes are highly compact, each with a total size of 24.2xa0Mb, and contain less than 2xa0% repetitive DNA. Clustering of genome-wide single nucleotide polymorphisms (SNP) of Pb3, Pb6 and three additional re-sequenced pathotypes (Pb2, Pb5 and Pb8) shows a high degree of correlation of cluster grouping with host range. The Pb3 genome features significant reduction of intergenic space with multiple examples of overlapping untranslated regions (UTRs). Dependency on the host for essential nutrients is evident from the loss of genes for the biosynthesis of thiamine and some amino acids and the presence of a wide range of transport proteins, including some unique to P. brassicae. The annotated genes of Pb3 include those with a potential role in the regulation of the plant growth hormones cytokinin and auxin. The expression profile of Pb3 genes, including putative effectors, during infection and their potential role in manipulation of host defence is discussed.ConclusionThe P. brassicae genome sequence reveals a compact genome, a dependency of the pathogen on its host for some essential nutrients and a potential role in the regulation of host plant cytokinin and auxin. Genome annotation supported by RNA sequencing reveals significant reduction in intergenic space which, in addition to low repeat content, has likely contributed to the P. brassicae compact genome.

Using Crude Whole-Genome Assemblies of Neisseria gonorrhoeae as a Platform for Strain Analysis: Clonal Spread of Gonorrhea Infection in Saskatchewan, Canada

ABSTRACT Using crude whole-genome assemblies, we analyzed 25 isolates of Neisseria gonorrhoeae by using a high-resolution single nucleotide polymorphism (SNP) approach for nine housekeeping genes, characterizing penA alleles, and antimicrobial susceptibility phenotypes coupled with population structure analysis. Two clonal complexes, characterized by their spatial and geographical persistence, were identified. In addition, the clonal spread of penicillin-resistant/intermediate phenotypes and a novel introduction of the azithromycin resistance phenotype in Saskatchewan, Canada, were ascertained using this method.

Brassica villosa , a system for studying non-glandular trichomes and genes in the Brassicas

Brassica villosa is a wild Brassica C genome species with very dense trichome coverage and strong resistance to many insect pests of Brassica oilseeds and vegetables. Transcriptome analysis of hairy B. villosa leaves indicated higher expression of several important trichome initiation genes compared with glabrous B. napus leaves and consistent with the Arabidopsis model of trichome development. However, transcripts of the TRY inhibitory gene in hairy B. villosa were surprisingly high relative to B. napus and relative transcript levels of SAD2, EGL3, and several XIX genes were low, suggesting potential ancillary or less important trichome-related roles for these genes in Brassica species compared with Arabidopsis. Several antioxidant, calcium, non-calcium metal and secondary metabolite genes also showed differential expression between these two species. These coincided with accumulation of two alkaloid-like compounds, high levels of calcium, and other metals in B. villosa trichomes that are correlated with the known tolerance of B. villosa to high salt and the calcium-rich natural habitat of this wild species. This first time report on the isolation of large amounts of pure B. villosa trichomes, on trichome content, and on relative gene expression differences in an exceptionally hairy Brassica species compared with a glabrous species opens doors for the scientific community to understand trichome gene function in the Brassicas and highlights the potential of B. villosa as a trichome research platform.

Hairy Canola (Brasssica napus) re-visited: Down-regulating TTG1 in an AtGL3-enhanced hairy leaf background improves growth, leaf trichome coverage, and metabolite gene expression diversity

BackgroundThrough evolution, some plants have developed natural resistance to insects by having hairs (trichomes) on leaves and other tissues. The hairy trait has been neglected in Brassica breeding programs, which mainly focus on disease resistance, yield, and overall crop productivity. In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), activates trichome initiation and patterning. Introduction of a trichome regulatory gene AtGL3 from Arabidopsis into semi-glabrous Brassica napus resulted in hairy canola plants which showed tolerance to flea beetles and diamondback moths; however plant growth was negatively affected. In addition, the role of BnTTG1 transcription in the new germplasm was not understood.ResultsHere, we show that two ultra-hairy lines (K-5-8 and K-6-3) with BnTTG1 knock-down in the hairy AtGL3+ B. napus background showed stable enhancement of trichome coverage, density, and length and restored wild type growth similar to growth of the semi-glabrous Westar plant. In contrast, over-expression of BnTTG1 in the hairy AtGL3+ B. napus background gave consistently glabrous plants of very low fertility and poor stability, with only one glabrous plant (O-3-7) surviving to the T3 generation. Q-PCR trichome gene expression data in leaf samples combining several leaf stages for these lines suggested that BnGL2 controlled B. napus trichome length and out-growth and that strong BnTTG1 transcription together with strong GL3 expression inhibited this process. Weak expression of BnTRY in both glabrous and trichome-bearing leaves of B. napus in the latter Q-PCR experiment suggested that TRY may have functions other than as an inhibitor of trichome initiation in the Brassicas. A role for BnTTG1 in the lateral inhibition of trichome formation in neighbouring cells was also proposed for B. napus. RNA sequencing of first leaves identified a much larger array of genes with altered expression patterns in the K-5-8 line compared to the hairy AtGL3+B. napus background (relative to the Westar control plant). These genes particularly included transcription factors, protein degradation and modification genes, but also included pathways that coded for anthocyanins, flavonols, terpenes, glucosinolates, alkaloids, shikimates, cell wall biosynthesis, and hormones. A 2nd Q-PCR experiment was conducted on redox, cell wall carbohydrate, lignin, and trichome genes using young first leaves, including T4 O-3-7-5 plants that had partially reverted to yield two linked growth and trichome phenotypes. Most of the trichome genes tested showed to be consistant with leaf trichome phenotypes and with RNA sequencing data in three of the lines. Two redox genes showed highest overall expression in K-5-8 leaves and lowest in O-3-7-5 leaves, while one redox gene and three cell wall genes were consistently higher in the two less robust lines compared with the two robust lines.ConclusionThe data support the strong impact of BnTTG1 knockdown (in the presence of strong AtGL3 expression) at restoring growth, enhancing trichome coverage and length, and enhancing expression and diversity of growth, metabolic, and anti-oxidant genes important for stress tolerance and plant health in B. napus. Our data also suggests that the combination of strong (up-regulated) BnTTG1 expression in concert with strong AtGL3 expression is unstable and lethal to the plant.

A landscape of hairy and twisted: hunting for new trichome mutants in the Saskatoon Arabidopsis T-DNA population

A total of 88 new Arabidopsis lines with trichome variation were recovered by screening 49,200 single-seed descent T3 lines from the SK activation-tagged population and from a new 20,000-line T-DNA insertion population (called pAG). Trichome variant lines were classified into 12 distinct phenotype categories. Single or multiple T-DNA insertion sites were identified for 89% of these mutant lines. Alleles of the well-known trichome genes TRY, GL2 and TTG1 were recovered with atypical phenotype variation not reported previously. Moreover, atypical gene expression profiles were documented for two additional mutants specifying TRY and GL2 disruptions. In remaining mutants, ten lines were disrupted in genes coding for proteins not implicated in trichome development, five were disrupted in hypothetical proteins and 11 were disrupted in proteins with unknown function. The collection represents new opportunities for the plant biology community to define trichome development more precisely and to refine the function of individual trichome genes.

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes.

Comparison of Five Major Trichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae

Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae.

A WD-repeat gene from peach (Prunus persica L.) is a functional ortholog of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1

We have cloned a WD-repeat gene from peach. The cloned gene is more than 3xa0kb and contains signature domains characteristic of WD-repeat genes. Because of its high homology with AtTTG1, we hypothesized that this gene could be a TTG1 ortholog in peach. Functional studies were carried out by complementing the trichome minus Arabidopsis ttg1-1 mutant with the putative peach TTG1 homolog. Successful restoration of normal trichomes was achieved in the resulting transgenics. We further tested the possibility that this gene was the candidate gene differentiating peach and nectarine. Sequence analysis indicated no difference in the full-length TTG1 and 1,600xa0bp of its promoter between peach and nectarine.