Putative Defense Genes Research Articles

Dual RNA-sequencing of Fusarium head blight resistance in winter wheat.

Fusarium head blight (FHB) is a devastating fungal disease responsible for significant yield losses in wheat and other cereal crops across the globe. FHB infection of wheat spikes results in grain contamination with mycotoxins, reducing both grain quality and yield. Breeding strategies have resulted in the production of FHB-resistant cultivars, however, the underlying molecular mechanisms of resistance in the majority of these cultivars are still poorly understood. To improve our understanding of FHB-resistance, we performed a transcriptomic analysis of FHB-resistant AC Emerson, FHB-moderately resistant AC Morley, and FHB-susceptible CDC Falcon in response to Fusarium graminearum. Wheat spikelets located directly below the point of inoculation were collected at 7-days post inoculation (dpi), where dual RNA-sequencing was performed to explore differential expression patterns between wheat cultivars in addition to the challenging pathogen. Differential expression analysis revealed distinct defense responses within FHB-resistant cultivars including the enrichment of physical defense through the lignin biosynthesis pathway, and DON detoxification through the activity of UDP-glycosyltransferases. Nucleotide sequence variants were also identified broadly between these cultivars with several variants being identified within differentially expressed putative defense genes. Further, F. graminearum demonstrated differential expression of mycotoxin biosynthesis pathways during infection, leading to the identification of putative pathogenicity factors.

Kiwifruit Resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. actinidiae and Defence Induction by Acibenzolar-S-methyl and Methyl Jasmonate Are Cultivar Dependent.

Pathogen susceptibility and defence gene inducibility were compared between the Actinidia arguta cultivar 'Hortgem Tahi' and the two cultivars of A. chinensis 'Hayward' and 'Zesy002'. Plants were treated with acibenzolar-s-methyl (ASM) or methyl jasmonate (MeJA) one week before inoculation with Pseudomonas syringae pv. actinidiae (Psa biovar3) or Sclerotinia sclerotiorum, or secondary induction with chitosan+glucan (Ch-Glu) as a potential pathogen proxy. Defence expression was evaluated by measuring the expression of 18 putative defence genes. 'Hortgem Tahi' was highly susceptible to sclerotinia and very resistant to Psa, whereas 'Zesy002' was highly resistant to both, and 'Hayward' was moderately susceptible to both. Gene expression in 'Hayward' and 'Zesy002' was alike but differed significantly from 'Hortgem Tahi' which had higher basal levels of PR1-i, PR5-i, JIH1, NPR3 and WRKY70 but lower expression of RD22 and PR2-i. Treatment with ASM caused upregulation of NIMIN2, PR1-i, WRKY70, DMR6 and PR5-i in all cultivars and induced resistance to Psa in 'Zesy002' and 'Hayward' but decreased resistance to sclerotinia in 'Zesy002'. MeJA application caused upregulation of LOX2 and downregulation of NIMIN2, DMR6 and PR2-i but did not affect disease susceptibility. The Ch-Glu inducer induced PR-gene families in each cultivar, highlighting its possible effectiveness as an alternative to actual pathogen inoculation. The significance of variations in fundamental and inducible gene expression among the cultivars is explored.

The Disease Progression and Molecular Defense Response in Chenopodium Quinoa Infected with Peronospora Variabilis, the Causal Agent of Quinoa Downy Mildew.

Downy mildew disease, caused by the biotrophic oomycete Peronospora variabilis, is the largest threat to the cultivation of quinoa (Chenopodium quinoa Willd.) in the Andean highlands, and occurs worldwide. However, so far, no molecular study of the quinoa-Peronospora interaction has been reported. Here, we developed tools to study downy mildew disease in quinoa at the gene expression level. P. variabilis was isolated and maintained, allowing the study of downy mildew disease progression in two quinoa cultivars under controlled conditions. Quinoa gene expression changes induced by P. variabilis were analyzed by qRT-PCR, for quinoa homologues of A. thaliana pathogen-associated genes. Overall, we observed a slower disease progression and higher tolerance in the quinoa cultivar Kurmi than in the cultivar Maniqueña Real. The quinoa orthologs of putative defense genes such as the catalase CqCAT2 and the endochitinase CqEP3 showed no changes in gene expression. In contrast, quinoa orthologs of other defense response genes such as the transcription factor CqWRKY33 and the chaperone CqHSP90 were significantly induced in plants infected with P. variabilis. These genes could be used as defense response markers to select quinoa cultivars that are more tolerant to P. variabilis infection.

Tissue-specific mRNA profiling of the Brassica napus-Sclerotinia sclerotiorum interaction uncovers novel regulators of plant immunity.

White mold is caused by the fungal pathogen Sclerotinia sclerotiorum and leads to rapid and significant loss in plant yield. Among its many brassicaceous hosts, including Brassica napus (canola) and Arabidopsis, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll, and vascular leaf tissue layers of B. napus in response to S. sclerotiorum. High-throughput tissue-specific mRNA sequencing increased the total number of detected transcripts compared with whole-leaf assessments and provided novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. When subjected to pathogen infection, the epidermis, mesophyll, and vasculature activate both specific and shared gene sets. Putative defense genes identified through transcription factor network analysis were then screened for susceptibility against necrotrophic, hemi-biotrophic, and biotrophic pathogens. Arabidopsis deficient in PR5-like RECEPTOR KINASE (PR5K) mRNA levels were universally susceptible to all pathogens tested and were further characterized to identify putative interacting partners involved in the PR5K signaling pathway. Together, these data provide insight into the complexity of the plant defense response directly at the site of infection.

Identification of Differentially Expressed Genes in Resistant Tetraploid Wheat (Triticum turgidum) under Sitobion avenae (F.) Infestation.

The grain aphid Sitobion avenae (Fabricius) is one of the most destructive pests of wheat (Triticum aestivum). Deployment of resistant wheat germplasm appears as an excellent solution for this problem. Elite bread wheat cultivars only have limited resistance to this pest. The present study was carried out to investigate the potential of the tetraploid wheat (Triticum turgidum) variety Lanmai, which showed high resistance to S. avenae at both seedling and adult plant stages, as a source of resistance genes. Based on apterous adult aphids’ fecundity tests and choice bioassays, Lanmai has been shown to display antixenosis and antibiosis. Suppression subtractive hybridization (SSH) was employed to identify and isolate the putative candidate defense genes in Lanmai against S. avenae infestation. A total of 134 expressed sequence tags (ESTs) were identified and categorized based on their putative functions. RT-qPCR analysis of 30 selected genes confirmed their differential expression over time between the resistant wheat variety Lanmai and susceptible wheat variety Polan305 during S. avenae infestation. There were 11 genes related to the photosynthesis process, and only 3 genes showed higher expression in Lanmai than in Polan305 after S. avenae infestation. Gene expression analysis also revealed that Lanmai played a critical role in salicylic acid and jasmonic acid pathways after S. avenae infestation. This study provided further insights into the role of defense signaling networks in wheat resistance to S. avenae and indicates that the resistant tetraploid wheat variety Lanmai may provide a valuable resource for aphid tolerance improvement in wheat.

Fruit Fly Larval Survival in Picked and Unpicked Tomato Fruit of Differing Ripeness and Associated Gene Expression Patterns.

Simple SummaryTephritid fruit flies are major pests to a wide range of fruits and vegetables. Female flies lay their eggs into the fruit where the resultant larvae cause damage and yield loss. To replace pesticide-based controls with more sustainable management approaches, we need to develop new generation technologies. Enhancing fruit resistance is a promising alternative but it has received limited research attention. In this study, we examined larval survival and gene expression changes of B. tryoni larvae and tomato fruit while the fruits were in different picking statuses (unpicked vs. picked) and ripening stages (colour break vs. fully ripe). We assessed larval survival in two time points of 48 h and 120 h after inoculation. The fruit picking status and ripening stage had a significant effect on B. tryoni larval survival at 120 h. The gene expression patterns were not affected by picking status; however, insect detoxification genes and plant-induced defence genes were upregulated across the treatments. Overall, we anticipated the lack of conformity between larval survival and gene expression as a result of overlooked candidate genes or critical sampling time points.The larvae of frugivorous tephritid fruit flies feed within fruit and are global pests of horticulture. With the reduced use of pesticides, alternative control methods are needed, of which fruit resistance is one. In the current study, we explicitly tested for phenotypic evidence of induced fruit defences by running concurrent larval survival experiments with fruit on or off the plant, assuming that defence induction would be stopped or reduced by fruit picking. This was accompanied by RT-qPCR analysis of fruit defence and insect detoxification gene expression. Our fruit treatments were picking status (unpicked vs. picked) and ripening stage (colour break vs. fully ripe), our fruit fly was the polyphagous Bactrocera tryoni, and larval survival was assessed through destructive fruit sampling at 48 and 120 h, respectively. The gene expression study targeted larval and fruit tissue samples collected at 48 h and 120 h from picked and unpicked colour-break fruit. At 120 h in colour-break fruit, larval survival was significantly higher in the picked versus unpicked fruit. The gene expression patterns in larval and plant tissue were not affected by picking status, but many putative plant defence and insect detoxification genes were upregulated across the treatments. The larval survival results strongly infer an induced defence mechanism in colour-break tomato fruit that is stronger/faster in unpicked fruits; however, the gene expression patterns failed to provide the same clear-cut treatment effect. The lack of conformity between these results could be related to expression changes in unsampled candidate genes, or due to critical changes in gene expression that occurred during the unsampled periods.

Transcriptomic Analysis of Quinoa Reveals a Group of Germin-Like Proteins Induced by Trichoderma.

Symbiotic strains of fungi in the genus Trichoderma affect growth and pathogen resistance of many plant species, but the interaction is not known in molecular detail. Here we describe the transcriptomic response of two cultivars of the crop Chenopodium quinoa to axenic co-cultivation with Trichoderma harzianum BOL-12 and Trichoderma afroharzianum T22. The response of C. quinoa roots to BOL-12 and T22 in the early phases of interaction was studied by RNA sequencing and RT-qPCR verification. Interaction with the two fungal strains induced partially overlapping gene expression responses. Comparing the two plant genotypes, a broad spectrum of putative quinoa defense genes were found activated in the cultivar Kurmi but not in the Real cultivar. In cultivar Kurmi, relatively small effects were observed for classical pathogen response pathways but instead a C. quinoa-specific clade of germin-like genes were activated. Germin-like genes were found to be more rapidly induced in cultivar Kurmi as compared to Real. The same germin-like genes were found to also be upregulated systemically in the leaves. No strong correlation was observed between any of the known hormone-mediated defense response pathways and any of the quinoa-Trichoderma interactions. The differences in responses are relevant for the capabilities of applying Trichoderma agents for crop protection of different cultivars of C. quinoa.

Expression of Putative Defense Responses in Cannabis Primed by Pseudomonas and/or Bacillus Strains and Infected by Botrytis cinerea.

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea’s infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.

Isolation of putative pepper defense-related genes against the pathogen Phytophthora capsici using suppression subtractive hybridization/macroarray and RNA-sequencing analyses

The oomycete Phytophthora capsici Leonian is one of the most economically important pathogens limiting pepper (Capsicum annuum) production in many regions around the world. Insights into the defense mechanisms of pepper against P. capsici would be helpful in the breeding of resistant cultivars. However, little is still known about the defense system in pepper against P. capsici. We used suppression subtractive hybridization (SSH) followed by macro-array screening to isolate the putative candidate defense genes (PSH, Phytophthora Subtractive Hybridization) in pepper that are differentially expressed between the resistant cultivar (CM334) and the susceptible cultivar (Chilsungcho) following P. capsici infection. A total of 72 PSH genes were identified and categorized based on their putative functions. Semi-quantitative RT-PCR analyses using 11 selected genes confirmed their differential expressions between the resistant and susceptible cultivars along the time course of infection with P. capsici. Furthermore, RNA-seq analyses were performed to understand the possible roles of PSH genes in the defense response to P. capsici as well as three viruses, including two tobacco mosaic virus strains and one Pepper Mottle Virus strain. We found that 37 genes out of 72 displayed differential expression in our RNA-seq-based heatmap between ‘CM334’ and ‘Chilsungcho’ upon pathogen infection. In particular, two genes, CA00g99220 and CA00g96010, and one gene, CA12g16620, were shown to be strongly and uniquely expressed in the resistant cultivar, CM334, against P. capsici and two viruses, respectively. Thus, we consider that this combined approach using SSH/macro-array screening and RNA-seq analyses is a relevant tool for isolation of candidate defense-related genes upon pathogen infection. Data in this study provide a good source for further study on the defense mechanisms against pathogens in chili pepper.

Genomic, morphological and functional characterisation of novel bacteriophage FNU1 capable of disrupting Fusobacterium nucleatum biofilms

Fusobacterium nucleatum is an important oral bacterium that has been linked to the development of chronic diseases such as periodontitis and colorectal cancer. In periodontal disease, F. nucleatum forms the backbone of the polymicrobial biofilm and in colorectal cancer is implicated in aetiology, metastasis and chemotherapy resistance. The control of this bacteria may be important in assisting treatment of these diseases. With increased rates of antibiotic resistance globally, there is need for development of alternatives such as bacteriophages, which may complement existing therapies. Here we describe the morphology, genomics and functional characteristics of FNU1, a novel bacteriophage lytic against F. nucleatum. Transmission electron microscopy revealed FNU1 to be a large Siphoviridae virus with capsid diameter of 88 nm and tail of approximately 310 nm in length. Its genome was 130914 bp, with six tRNAs, and 8% of its ORFs encoding putative defence genes. FNU1 was able to kill cells within and significantly reduce F. nucleatum biofilm mass. The identification and characterisation of this bacteriophage will enable new possibilities for the treatment and prevention of F. nucleatum associated diseases to be explored.

In silico prediction and segregation analysis of putative virus defense genes based on SSR markers in sweet potato F1 progenies of cultivars ‘New Kawogo’ and ‘Resisto’

In sweet potato, an anti-virus defense mechanism termed reversion has been postulated to lead to virus freedom from once infected plants. The objectives of this study were to identify anti-virus defense genes and evaluate their segregation in progenies. Reference genes from different plant species were used to assemble transcript sequences of each sweet potato defense gene in silico. Sequences were used for evaluate phylogenetic relationships with similar genes from different plant species, mining respective defense genes and thereafter developing simple sequence repeats (SSRs) for segregation analysis. Eight potential defense genes were identified: RNA dependent RNA polymerases 1, 2, 5, and 6; Argonaute 1, and Dicer-like 1, 2, and 4. Identified genes were differentially related to those of other plants and were observed on different chromosomes. The defense genes contained mono-, di-, tri-, tetra, penta-, and hexa-nucleotide repeat motifs. The SSR markers within progenies were segregated in disomic, co-segregation, nullisomic, monosomic, and trisomic modes. These findings indicate the possibility of deriving and utilizing SSRs using published genomic information. Furthermore, and given that the SSR markers were derived from known genes on defined chromosomes, this work will contribute to future molecular breeding and development of resistance gene analogs in this economically important crop.

Elicitor induction of defence genes and reduction of bacterial canker in kiwifruit

Pseudomonas syringae pv. actinidiae (Psa), which causes bacterial canker, is the most serious global pathogen of kiwifruit. Like most bacterial pathogens, control options are limited, but elicitors can reduce disease significantly, particularly those that induce the salicylic acid (SA) pathway. Acibenzolar-S-methyl (ASM), a SA analogue, is one of the most effective elicitors for Psa control. In this study, real-time PCR (qPCR) was used to measure the expression of 18 putative defence genes in Actinidia chinensis var. chinensis ‘Hort16A’ in response to Psa and ASM. Application of ASM led to up-regulation of RPM1 interacting protein 4 (RIN4), phenylalanine ammonia lyase (PAL), a hypersensitivity-induced response protein (HIRP), and β-1,3-glucosidase. Expression of PAL and HIRP was further enhanced when elicitor application and Psa-inoculation were combined. Elevated gene expression was correlated with decreased disease expression, and supports the hypothesis that elicitor-treated plants are primed to react more rapidly and/or strongly to pathogens.

Tanscriptomic Study of the Soybean-Fusarium virguliforme Interaction Revealed a Novel Ankyrin-Repeat Containing Defense Gene, Expression of Whose during Infection Led to Enhanced Resistance to the Fungal Pathogen in Transgenic Soybean Plants.

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.

Identification of defense-related genes in banana roots infected by Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium oxysporum f. sp. cubense tropical race 4 is a lethal pathogen, affecting banana production by causing vascular wilt. To date, the molecular basis of resistant cultivars is largely unknown. In this study, suppression subtractive hybridization (SSH) libraries were constructed for the roots of two iso-line cultivars—a Foc-susceptible cultivar and its tolerant mutant—using both uninfected controls and plants artificially challenged with pathogen conidiospores. Unigenes from SSH libraries were used to develop a cDNA array, and then microarray analysis was carried out to verify those genes exhibiting differential expression. A total of 257 unigenes was obtained from the SSH libraries and microarray detection confirmed that most of them were differentially expressed. Of these, 116 unigenes had matched proteins in gene ontology and were classified into 38 different functional groups. The transcript levels of up to 19 putative defense genes were followed for a whole infection period in four cultivars differing in resistance level. At the early stages of infection (within 48 h), nineteen genes varied by a factor of more than three, subsequently only four genes (Pectinesterase inhibitor, PEI; Peroxidase, POD; Pathogenesis-related protein 1, PR1; and Pathogenesis-related protein 3, PR3) maintained their up-regulation. This indicates that anti-oxidation, cell wall modification and synthesis of anti-fungal proteins are the most important aspects to study if we are to understand the Foc resistance mechanism in commercial banana plants.

Characterisation, evolutionary trends and mapping of putative resistance and defence genes in Actinidia (kiwifruit)

A virulent strain of Pseudomonas syringae actinidae has invaded kiwifruit (genus Actinidia) orchards in New Zealand, making the study of Actinidia resistance genes critical. Examination of expressed sequence tag (EST) libraries constructed in various species of Actinidia showed that, based on homologies to Arabidopsis, many families of resistance genes were represented. The mapping of these genes to establish their chromosomal positions on the Actinidia chinensis genetic linkage map was facilitated by designing 71 primer pairs to the EST sequences. The genetic markers mapped across 22 of the linkage groups, both as individuals and as members of a cluster. The clusters were generally homologous in nature, though heterologous examples were present. A sample of genes representing both the basal defence and the resistance-gene mediated defence pathways, here termed collectively ‘resistance genes’, was characterised. Chromosomal sites of resistance gene markers representing a range of such genes have been identified and can be considered during the selection of parents in breeding programmes.

Transcriptome and metabolite profiling of the infection cycle of Zymoseptoria tritici on wheat reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions and a variation on the hemibiotrophic lifestyle definition.

The hemibiotrophic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat (Triticum aestivum). Pathogen reproduction on wheat occurs without cell penetration, suggesting that dynamic and intimate intercellular communication occurs between fungus and plant throughout the disease cycle. We used deep RNA sequencing and metabolomics to investigate the physiology of plant and pathogen throughout an asexual reproductive cycle of Z. tritici on wheat leaves. Over 3,000 pathogen genes, more than 7,000 wheat genes, and more than 300 metabolites were differentially regulated. Intriguingly, individual fungal chromosomes contributed unequally to the overall gene expression changes. Early transcriptional down-regulation of putative host defense genes was detected in inoculated leaves. There was little evidence for fungal nutrient acquisition from the plant throughout symptomless colonization by Z. tritici, which may instead be utilizing lipid and fatty acid stores for growth. However, the fungus then subsequently manipulated specific plant carbohydrates, including fructan metabolites, during the switch to necrotrophic growth and reproduction. This switch coincided with increased expression of jasmonic acid biosynthesis genes and large-scale activation of other plant defense responses. Fungal genes encoding putative secondary metabolite clusters and secreted effector proteins were identified with distinct infection phase-specific expression patterns, although functional analysis suggested that many have overlapping/redundant functions in virulence. The pathogenic lifestyle of Z. tritici on wheat revealed through this study, involving initial defense suppression by a slow-growing extracellular and nutritionally limited pathogen followed by defense (hyper) activation during reproduction, reveals a subtle modification of the conceptual definition of hemibiotrophic plant infection.

RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug

BackgroundBed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.ResultsWe performed a next-generation RNA sequencing (RNA-Seq) experiment to find differentially expressed genes between pesticide-resistant (PR) and pesticide-susceptible (PS) strains of C. lectularius. A reference transcriptome database of 51,492 expressed sequence tags (ESTs) was created by combining the databases derived from de novo assembled mRNA-Seq tags (30,404 ESTs) and our previous 454 pyrosequenced database (21,088 ESTs). The two-way GLMseq analysis revealed ~15,000 highly significant differentially expressed ESTs between the PR and PS strains. Among the top 5,000 differentially expressed ESTs, 109 putative defense genes (cuticular proteins, cytochrome P450s, antioxidant genes, ABC transporters, glutathione S-transferases, carboxylesterases and acetyl cholinesterase) involved in penetration resistance and metabolic resistance were identified. Tissue and development-specific expression of P450 CYP3 clan members showed high mRNA levels in the cuticle, Malpighian tubules, and midgut; and in early instar nymphs, respectively. Lastly, molecular modeling and docking of a candidate cytochrome P450 (CYP397A1V2) revealed the flexibility of the deduced protein to metabolize a broad range of insecticide substrates including DDT, deltamethrin, permethrin, and imidacloprid.ConclusionsWe developed significant molecular resources for C. lectularius putatively involved in metabolic resistance as well as those participating in other modes of insecticide resistance. RNA-Seq profiles of PR strains combined with tissue-specific profiles and molecular docking revealed multi-level insecticide resistance in C. lectularius. Future research that is targeted towards RNA interference (RNAi) on the identified metabolic targets such as cytochrome P450s and cuticular proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in C. lectularius.

Down Regulation of Putative Defence-associated Transcripts Correlates with Ripe Rot Symptoms on Kiwifruit (Actinidia chinensis)

A real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was employed to investigate the transcriptional levels of putative defence genes expressed during postharvest storage of Actinidia chinensis‘Hort16A’ kiwifruit. Significant decreases (80% reduction) in normalized gene expression over time relative to the basal levels of gene expression at harvest in control fruit were observed for thaumatin-like protein (TLP), class IV acidic chitinase, chalcone-flavonone isomerase (CHI) and glucan endo-1,3-beta-glucosidase (β-1,3-glucosidase). Reduction in transcript abundance for these genes paralleled the significant increase in postharvest ripe rot disease incidence over time (P = 0.0008). Gene expression levels were approximately the same in control vs. inoculated fruit for all the four genes described above, except for β-1,3-glucosidase where expression was significantly greater (P = 0.007) in inoculated than in control fruit. For one of the genes of interest that we had studied by qRT-PCR, TLP, a small amount of protein was purified and assayed in vitro for activity against Cryptosporiopsis actinidiae and Phomopsis spp., the causal agents of ripe rots of ‘Hort16A’ kiwifruit. TLP from kiwifruit did not appear to be directly toxic to either pathogen. TLP does not prevent ripe rots, but it may be useful as a marker of resistance given the temporal correlation between decreased TLP and increased ripe rots that was demonstrated by qRT-PCR.

Transcriptomics of the bed bug (Cimex lectularius).

BackgroundBed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance.Methodology and Principal FindingsUsing 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database.ConclusionsTo our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies.

Identification of differentially expressed genes in a resistant versus a susceptible blueberry cultivar after infection by Colletotrichum acutatum.

Anthracnose fruit rot, caused by the fungus Colletotrichum acutatum, is an important disease of blueberry worldwide. The cultivar Elliott is resistant, severely restricting fungal growth and sporulation relative to the susceptible cultivar Jersey. The objectives of this research were as follows: (i) to analyse pre-penetration events in 'Elliott' and 'Jersey' at different stages of fruit development; (ii) to identify putative defence genes in 'Elliott' fruit; and (iii) to monitor the timing of the oxidative burst in both cultivars. Light microscopy revealed no differences in the pre-penetration activities of C. acutatum on the immature fruit of both cultivars. However, at fruit ripening, conidia germinated and formed appressoria more rapidly on 'Jersey' than on 'Elliott' fruit. Using suppression subtractive hybridization, 37 differentially expressed sequence tags (ESTs) were detected in 'Elliott' versus 'Jersey' upon infection. Several of the ESTs had homology to known plant defence genes, such as a class II chitinase, pathogenesis-related protein 10 (PR10) and β-1,3-glucanase. Two putative genes involved in oxidative stress were identified: a metallothionein-like protein and monodehydroascorbate reductase. ESTs of fungal origin were also detected. Many ESTs had no homology to known genes. Using semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR), the expression of most of the candidate genes was detected earlier in 'Elliott' than in 'Jersey', some within 24 h post inoculation (hpi). Monitoring of the oxidative burst showed that the overall H(2) O(2) concentration was two to three times higher in 'Elliott' than in 'Jersey' at 24 hpi. The elucidation of the basis of resistance to C. acutatum in blueberry will facilitate the development of new anthracnose fruit rot-resistant cultivars.