Costs of resistance and infection by a generalist pathogen

Pathogen infection is typically costly to hosts, resulting in reduced fitness. However, pathogen exposure may also come at a cost even if the host does not become infected. These fitness reductions, referred to as “resistance costs”, are inducible physiological costs expressed as a result of a trade‐off between resistance to a pathogen and aspects of host fitness (e.g., reproduction). Here, we examine resistance and infection costs of a generalist fungal pathogen (Metschnikowia bicuspidata) capable of infecting a number of host species. Costs were quantified as reductions in host lifespan, total reproduction, and mean clutch size as a function of pathogen exposure (resistance cost) or infection (infection cost). We provide empirical support for infection costs and modest support for resistance costs for five Daphnia host species. Specifically, only one host species examined incurred a significant cost of resistance. This species was the least susceptible to infection, suggesting the possibility that host susceptibility to infection is associated with the detectability and size of resistance cost. Host age at the time of pathogen exposure did not influence the magnitude of resistance or infection cost. Lastly, resistant hosts had fitness values intermediate between unexposed control hosts and infected hosts. Although not statistically significant, this could suggest that pathogen exposure does come at some marginal cost. Taken together, our findings suggest that infection is costly, resistance costs may simply be difficult to detect, and the magnitude of resistance cost may vary among host species as a result of host life history or susceptibility.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Quantitative trait loci affecting morphology traits in gilthead seabream (Sparus aurata L.)

We report a quantitative trait loci (QTL) mapping study on 18 morphometric characters in gilthead seabream based on a total of 74 informative microsatellite markers genotyped in 409 offspring coming from 10 paternal half‐sib families. Statistical analysis was carried out using a linear regression approach, and various suggestive and significant morphology QTL were detected in three (9, 21 and 25) of nine linkage groups examined. Fitting body weight as a covariate reduced the significance of some QTL but revealed three new QTL in other linkage groups (LG6 and LG10). Current results combined with those obtained from previous studies underline highly significant loci affecting overall growth and morphology in S. aurata.     

SNP and INDEL detection in a QTL region on chicken chromosome 2 associated with muscle deposition

Genetic improvement is important for the poultry industry, contributing to increased efficiency of meat production and quality. Because breast muscle is the most valuable part of the chicken carcass, knowledge of polymorphisms influencing this trait can help breeding programs. Therefore, the complete genome of 18 chickens from two different experimental lines (broiler and layer) from EMBRAPA was sequenced, and SNPs and INDELs were detected in a QTL region for breast muscle deposition on chicken chromosome 2 between microsatellite markers MCW0185 and MCW0264 (105 849–112 649 kb). Initially, 94 674 unique SNPs and 10 448 unique INDELs were identified in the target region. After quality filtration, 77% of the SNPs (85 765) and 60% of the INDELs (7828) were retained. The studied region contains 66 genes, and functional annotation of the filtered variants identified 517 SNPs and three INDELs in exonic regions. Of these, 357 SNPs were classified as synonymous, 153 as non‐synonymous, three as stopgain, four INDELs as frameshift and three INDELs as non‐frameshift. These exonic mutations were identified in 37 of the 66 genes from the target region, three of which are related to muscle development (DTNA, RB1CC1 and MOS). Fifteen non‐tolerated SNPs were detected in several genes (MEP1B, PRKDC, NSMAF, TRAPPC8, SDR16C5, CHD7, ST18 and RB1CC1). These loss‐of‐function and exonic variants present in genes related to muscle development can be considered candidate variants for further studies in chickens. Further association studies should be performed with these candidate mutations as should validation in commercial populations to allow a better explanation of QTL effects.     

Incapacitating effects of fungal coinfection in a novel pathogen system

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd‐susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community‐level declines.     

Variation in resistance to multiple pathogen species: anther smuts of Silene uniflora

The occurrence of multiple pathogen species on a shared host species is unexpected when they exploit the same micro‐niche within the host individual. One explanation for such observations is the presence of pathogen‐specific resistances segregating within the host population into sites that are differentially occupied by the competing pathogens. This study used experimental inoculations to test whether specific resistances may contribute to the maintenance of two species of anther‐smut fungi, Microbotryum silenes‐inflatae and Microbotryum lagerheimii, in natural populations of Silene uniflora in England and Wales. Overall, resistance to the two pathogens was strongly positively correlated among host populations and to a lesser degree among host families within populations. A few instances of specific resistance were also observed and confirmed by replicated inoculations. The results suggest that selection for resistance to one pathogen may protect the host from the emergence via host shifts of related pathogen species, and conversely that co‐occurrence of two species of pathogens may be dependent on the presence of host genotypes susceptible to both.     

Effect of a major QTL affecting IPN resistance on production traits in Atlantic salmon

This study investigated the effect of a major QTL for resistance to IPN in salmon on performance and production traits. The traits studied were related to growth, fillet and gutted yields, and fat content. Two different analyses were performed: (1) regression of the phenotypic data of the production traits on the predicted number of resistant IPN‐QTL alleles in individuals and (2) a variance component analysis using the (co)variance matrix calculated at the putative location of the QTL. No significant effect of the QTL was detected on any of the traits investigated by either method. The result has important practical implications in that it encourages the use of MAS to reduce the risks and impact of IPN mortality.     

Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome

Brassica oleracea is an important agricultural species encompassing many vegetable crops including cabbage, cauliflower, broccoli and kale; however, it can be susceptible to a variety of fungal diseases such as clubroot, blackleg, leaf spot and downy mildew. Resistance to these diseases is meditated by specific disease resistance genes analogs (RGAs) which are differently distributed across B. oleracea lines. The sequenced reference cultivar does not contain all B. oleracea genes due to gene presence/absence variation between individuals, which makes it necessary to search for RGA candidates in the B. oleracea pangenome. Here we present a comparative analysis of RGA candidates in the pangenome of B. oleracea. We show that the presence of RGA candidates differs between lines and suggests that in B. oleracea, SNPs and presence/absence variation drive RGA diversity using separate mechanisms. We identified 59 RGA candidates linked to Sclerotinia, clubroot, and Fusarium wilt resistance QTL, and these findings have implications for crop breeding in B. oleracea, which may also be applicable in other crops species.     

QTL analysis of resistance to the rice blast pathogen in barley (Hordeum vulgare)

Barley is compatible with the rice blast pathogen (Pyricularia oryzae Cav.). Fiftyfour barley cultivars of diverse geographic origin and pedigree were inoculated with three isolates of the rice blast pathogen. All barley genotypes showed blast disease symptoms when inoculated at the seedling stage with each of the three isolates. However, one genotype showed quantitative resistance to all three isolates and three genotypes showed quantitative resistance to one or two of the isolates. By inoculating a set of doubled-haploid lines derived from the cross ’Harrington’ (susceptible) and ’TR306’ (resistant) with isolate Ken 54–20, we mapped quantitative trait loci (QTLs) determining seedling stage blast resistance. At all QTLs, TR306 contributed the resistance alleles. The four QTLs, when considered jointly, explained 43.6% of the phenotypic variation in blast symptom expression. A comparison of the blast resistance QTLs with other disease resistance QTLs reported in this population revealed a region on chromosome 4 (4H) with multiple disease resistance loci. It will be useful to capitalize on the syntenic relationship of rice and barley and to integrate information on species-specific resistance genes with information on the reaction of the two species to the same pathogen. Received: 7 January 2000 / Accepted: 22 September 2000     

Host‐specific thermal profiles affect fitness of a widespread pathogen

Host behavior can interact with environmental context to influence outcomes of pathogen exposure and the impact of disease on species and populations. Determining whether the thermal behaviors of individual species influence susceptibility to disease can help enhance our ability to explain and predict how and when disease outbreaks are likely to occur. The widespread disease chytridiomycosis (caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd) often has species‐specific impacts on amphibian communities; some host species are asymptomatic, whereas others experience mass mortalities and population extirpation. We determined whether the average natural thermal regimes experienced by sympatric frog species in nature, in and of themselves, can account for differences in vulnerability to disease. We did this by growing Bd under temperatures mimicking those experienced by frogs in the wild. At low and high elevations, the rainforest frogs Litoria nannotis, L. rheocola, and L. serrata maintained mean thermal regimes within the optimal range for pathogen growth (15–25°C). Thermal regimes for L. serrata, which has recovered from Bd‐related declines, resulted in slower pathogen growth than the cooler and less variable thermal regimes for the other two species, which have experienced more long‐lasting declines. For L. rheocola and L. serrata, pathogen growth was faster in thermal regimes corresponding to high elevations than in those corresponding to low elevations, where temperatures were warmer. For L. nannotis, which prefers moist and thermally stable microenvironments, pathogen growth was fastest for low‐elevation thermal regimes. All of the thermal regimes we tested resulted in pathogen growth rates equivalent to, or significantly faster than, rates expected from constant‐temperature experiments. The effects of host body temperature on Bd can explain many of the broad ecological patterns of population declines in our focal species, via direct effects on pathogen fitness. Understanding the functional response of pathogens to conditions experienced by the host is important for determining the ecological drivers of disease outbreaks.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

Comparative study of sequence aligners for detecting antibiotic resistance in bacterial metagenomes

We aim to compare the performance of Bowtie2 , bwa‐mem , blastn and blastx when aligning bacterial metagenomes against the Comprehensive Antibiotic Resistance Database (CARD). Simulated reads were used to evaluate the performance of each aligner under the following four performance criteria: correctly mapped, false positives, multi‐reads and partials. The optimal alignment approach was applied to samples from two wastewater treatment plants to detect antibiotic resistance genes using next generation sequencing. blastn mapped with greater accuracy among the four sequence alignment approaches considered followed by Bowtie2 . blastx generated the greatest number of false positives and multi‐reads when aligned against the CARD. The performance of each alignment tool was also investigated using error‐free reads. Although each aligner mapped a greater number of error‐free reads as compared to Illumina‐error reads, in general, the introduction of sequencing errors had little effect on alignment results when aligning against the CARD. Given each performance criteria, blastn was found to be the most favourable alignment tool and was therefore used to assess resistance genes in sewage samples. Beta‐lactam and aminoglycoside were found to be the most abundant classes of antibiotic resistance genes in each sample.

Significance and Impact of the Study

Antibiotic resistance genes (ARGs) are pollutants known to persist in wastewater treatment plants among other environments, thus methods for detecting these genes have become increasingly relevant. Next generation sequencing has brought about a host of sequence alignment tools that provide a comprehensive look into antimicrobial resistance in environmental samples. However, standardizing practices in ARG metagenomic studies is challenging since results produced from alignment tools can vary significantly. Our study provides sequence alignment results of synthetic, and authentic bacterial metagenomes mapped against an ARG database using multiple alignment tools, and the best practice for detecting ARGs in environmental samples.     

Cloning and identification of the promoter of the tobacco Sar8.2b gene,a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as-1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.     

The role of pleiotropy and linkage in genes affecting a sexual ornament and bone allocation in the chicken

Sexual selection and the ornaments that inform such choices have been extensively studied, particularly from a phenotypic perspective. Although more is being revealed about the genetic architecture of sexual ornaments, much still remains to be discovered. The comb of the chicken is one of the most widely recognized sexual ornaments, which has been shown to be correlated with both fecundity and bone allocation. In this study, we use a combination of multiple intercrosses between White Leghorn populations and wild‐derived Red Junglefowl to, first, map quantitative trait loci (QTL) for bone allocation and, second, to identify expression QTL that correlate and colocalize with comb mass. These candidate quantitative genes were then assessed for potential pleiotropic effects on bone tissue and fecundity traits. We identify genes that correlate with both relative comb mass and bone traits suggesting a combination of both pleiotropy and linkage mediates gene regulatory variation in these traits.