Bart Lievens

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

Recent Developments in Pathogen Detection Arrays: Implications for Fungal Plant Pathogens and Use in Practice

ABSTRACT The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. Increasingly, diagnostic laboratories are pursuing fast routine methods that provide reliable identification, sensitive detection, and accurate quantification of plant pathogens. In addition, since plants or parts thereof can be infected by multiple pathogens, multiplex assays that can detect and quantify different pathogens simultaneously are highly desirable. Technologies that can meet these requirements, especially those involving polymerase chain reaction, are being developed and implemented in horticultural and agricultural practice. Currently, DNA array technology is the most suitable technique for multiplex detection of plant pathogens. Recently, a quantitative aspect was added to this technology, making DNA arrays highly attractive for various research and practical applications. Here, we review the most important recent advances in molecular plant pathogen diagnostics, with special attention to fungal molecular diagnostics. In addition to their applicability in practice, the different criteria that have to be fulfilled for developing robust detection procedures that can routinely be used by diagnostic laboratories are discussed.

Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum

Rapid and reliable detection and identification of potential plant pathogens is required for taking appropriate and timely disease management measures. For many microbial species of which all strains generally are plant pathogens on a known host range, this has become quite straightforward. However, for some fungal species this is quite a challenge. One of these is Fusarium oxysporum Schlechtend:Fr., which, as a species, has a very broad host range, while individual strains are usually highly host-specific. Moreover, many strains of this fungus are non-pathogenic soil inhabitants. Thus, with regard to effective disease management, identification below the species level is highly desirable. So far, the genetic basis of host specificity in F. oxysporum is poorly understood. Furthermore, strains that infect a particular plant species are not necessarily more closely related to each other than to strains that infect other hosts. Despite these difficulties, recently an increasing number of studies have reported the successful development of molecular markers to discriminate F. oxysporum strains below the species level.

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates.

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.

Analysis of network architecture reveals phylogenetic constraints on mycorrhizal specificity in the genus Orchis (Orchidaceae)

The specificity of orchids for their fungi can vary substantially, from highly specialist interactions to more generalist interactions, but little is known about the evolutionary history of the mycorrhizal specificity of orchids. Here, we used a network analysis approach to investigate orchid mycorrhizal associations in 16 species of the genus Orchis sampled across 11 different regions in Europe. We first examined in detail the structure of the network of associations and then tested for a phylogenetic signal in mycorrhizal specificity and identified the fungi with which the orchids associated. We found 20 different fungal lineages that associated with species of the genus Orchis, most of them being related to members of the Tulasnellaceae (84.33% of all identified associations) and a smaller proportion being related to members of the Ceratobasidiaceae (9.97%). Species associations formed a nested network that is built on asymmetric links among species. Evolution of mycorrhizal specificity in Orchis closely resembles a Brownian motion process, and the interaction between Orchis and Tulasnellaceae fungi is significantly influenced by the phylogenetic relationships between the Orchis species. Our results provide evidence of the presence of phylogenetic conservatism in mycorrhizal specificity in orchids and demonstrate that evolutionary processes may be an important factor in generating patterns of mycorrhizal associations.

Genome-Wide Characterization of ISR Induced in Arabidopsis thaliana by Trichoderma hamatum T382 Against Botrytis cinerea Infection

In this study, the molecular basis of the induced systemic resistance (ISR) in Arabidopsis thaliana by the biocontrol fungus Trichoderma hamatum T382 against the phytopathogen Botrytis cinerea B05-10 was unraveled by microarray analysis both before (ISR-prime) and after (ISR-boost) additional pathogen inoculation. The observed high numbers of differentially expressed genes allowed us to classify them according to the biological pathways in which they are involved. By focusing on pathways instead of genes, a holistic picture of the mechanisms underlying ISR emerged. In general, a close resemblance is observed between ISR-prime and systemic acquired resistance, the systemic defense response that is triggered in plants upon pathogen infection leading to increased resistance toward secondary infections. Treatment with T. hamatum T382 primes the plant (ISR-prime), resulting in an accelerated activation of the defense response against B. cinerea during ISR-boost and a subsequent moderation of the B. cinerea induced defense response. Microarray results were validated for representative genes by qRT-PCR. The involvement of various defense-related pathways was confirmed by phenotypic analysis of mutants affected in these pathways, thereby proving the validity of our approach. Combined with additional anthocyanin analysis data these results all point to the involvement of the phenylpropanoid pathway in T. hamatum T382-induced ISR.

Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness.

Differential Tomato Transcriptomic Responses Induced by Pepino Mosaic Virus Isolates with Differential Aggressiveness

Pepino mosaic virus (PepMV) is a highly infectious potexvirus and a major disease of greenhouse tomato (Solanum lycopersicum) crops worldwide. Damage and economic losses caused by PepMV vary greatly and can be attributed to differential symptomatology caused by different PepMV isolates. Here, we used a custom-designed Affymetrix tomato GeneChip array with probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation of tomato seedlings with a mild and an aggressive PepMV isolate that share 99.4% nucleotide sequence identity. The two isolates induced a different transcriptomic response, despite accumulating to similar viral titers. PepMV inoculation resulted in repression of photosynthesis. In addition, defense responses were stronger upon inoculation with the aggressive isolate, in both cases mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for a PepMV-encoded silencing suppressor. Finally, perturbation of pigment biosynthesis, as shown by differential regulation of the flavonoid and lycopene biosynthesis pathways, was monitored. Metabolite analyses on mature fruits of PepMV-infected tomato plants, which showed typical fruit marbling, revealed a decrease in carotenoids, likely responsible for the marbled phenotype, and an increase in alkaloids and phenylpropanoids that are associated with pathogen defense in the yellow sectors of the fruit.

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and in silico analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of in silico primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.

From extensive clone libraries to comprehensive DNA arrays for the efficient and simultaneous detection and identification of orchid mycorrhizal fungi.

A DNA array was developed from extensive clone library sequence data sets for the assessment of dominant members of mycorrhizal fungi that associate with terrestrial orchid species. As a-proof-of-concept, the array was developed for the basidiomycetous mycorrhizal partners from three closely related perennial Orchis species, including Orchis anthropophora, O. militaris and O. purpurea. Based on internal transcribed spacer regions, oligonucleotides were developed for seven operational taxonomic units (OTUs; defined as groups of sequences sharing at least 97% sequence similarity), corresponding to members of the Tulasnellaceae family. In order to cover a broader spectrum of tulasnelloid fungi, oligonucleotides were as well developed for two subsets of closely related OTUs. The array was evaluated using multiple primer pairs. In addition, hybridization results were validated by recovery and sequencing of the hybridized amplicons as well as by hybridizing reference DNA samples. Considering the unlimited expansion possibilities of DNA arrays to include specific detector oligonucleotides for other and more microorganisms, the method described here has the major advantage that it provides a powerful, rapid and cost-effective way for the simultaneous detection and identification of a wide range of orchid mycorrhizae. The design, development and advantages of the array are discussed in relation to its potential for future research in mycorrhizal ecology.