Mark S. Phillips

Efficient Virus-Induced Gene Silencing in Roots Using a Modified Tobacco Rattle Virus Vector

Due to their capability of eliciting a form of posttranscriptional gene silencing (termed virus-induced gene silencing or VIGS), plant viruses are increasingly used as reverse-genetics tools for functional characterization of plant genes. RNA viruses have been shown to trigger silencing in a variety of host plants, including members of Solanacae and Arabidopsis (Arabidopsis thaliana). Several factors affect the silencing response, including host range and viral tropism within the plant. The work presented here demonstrates that a modified tobacco rattle virus (TRV) vector retaining the helper protein 2b, required for transmission by a specific vector nematode, not only invades and replicates extensively in whole plants, including meristems, but also triggers a pervasive systemic VIGS response in the roots of Nicotiana benthamiana, Arabidopsis, and tomato (Lycopersicon esculentum). This sustained VIGS response was exemplified by the silencing of genes involved in root development (IRT1, TTG1 [transparent testa glabra], RHL1 [root hairless1], and β-tubulin), lateral root-meristem function (RML1 [root meristemless1]), and nematode resistance (Mi). Roots of silenced plants exhibit reduced levels of target mRNA and phenocopy previously described mutant alleles. The TRV-2b vector displays increased infectivity and meristem invasion, both key requirements for efficient VIGS-based functional characterization of genes in root tissues. Our data suggest that the TRV helper protein 2b may have an essential role in the host regulatory mechanisms that control TRV invasion.

Characterization of a chorismate mutase from the potato cyst nematode Globodera pallida

SUMMARY Some plant endoparasitic nematodes are biotrophic and induce remarkable changes in their hosts in order to ensure a continuous supply of food. Proteins secreted from oesophageal gland cells have been implicated in this pathogenic process. A potentially secreted chorismate mutase has been isolated from the potato cyst nematode Globodera pallida. The gene encoding this protein is expressed in the subventral oesophageal gland cells of the nematode, and the mRNA derived from this gene is only present in the early parasitic stages. Sequence analysis of this gene shows that, like other genes involved in the host-parasite interaction of plant parasitic nematodes, it is likely to have been acquired by horizontal gene transfer from bacteria. The presence of a signal peptide in the deduced amino acid sequence of the G. pallida chorismate mutase and its expression in the subventral oesophageal gland cells suggest that it is secreted from the nematode, pointing to a role for the protein in the host-parasite interaction. The shikimate pathway, of which chorismate mutase is normally a part, is not found in animals but is present in plants and bacteria. In plants it gives rise to a variety of compounds which are important in amino acid synthesis and defence signalling pathways, as well as auxins, which have been implicated in the early development of nematode feeding sites. The potential roles of a nematode chorismate mutase are discussed.

Identification of AFLP and SSR markers associated with quantitative resistance to Globodera pallida (Stone) in tetraploid potato (Solanum tuberosum subsp. tuberosum) with a view to marker-assisted selection

Abstract Seventy eight clones from the cross between SCRI clone 12601ab1 and cv Stirling were used to explore the possibility of genetical linkage analysis in tetraploid potato (Solanum tuberosum subsp. tuberosum). Clone 12601ab1 had quantitative resistance to Globodera pallida Pa2/3 derived from S. tuberosum subsp. andigena. The strategy adopted involved identifying single- (simplex) and double- (duplex) dose AFLP markers in the parents from segregation ratios that could be unambiguously identified in their offspring, detecting linkage between a marker and a putative quantitative trait locus (QTL) for resistance, and placing the QTL on the linkage map of markers. The numbers of scorable segregating markers were 162 simplex ones present only in 12601ab1, 87 present in Stirling, and 32 present in both; and 72 duplex markers present only in 12601ab1 and 45 present in Stirling. The total map length was 990.9 cM in 12601ab1 and 484.6 cM in Stirling. A QTL with a resistance allele present in double dose (QQqq) in 12601ab1 was inferred from the associations between resistance scores (square root of female counts) and two duplex markers linked in coupling, which, in turn, were linked in coupling to four simplex markers also associated with resistance, but to a lesser degree. The largest marker class difference was the one for the duplex marker P61M34=15. It accounted for 27.8% of the phenotypic variance in resistance scores, or approximately 30% of the genotypic variance. Subsequently, this duplex marker was found to be linked in coupling with a duplex SSR allele Stm3016=a, whose locus was shown to be on chromosome IV in a diploid reference mapping population. The other QTLs for resistance segregating in the progeny were not identified for one or more of the following reasons: the markers did not cover the whole of the genome, there were unfavourable repulsion linkages between the QTLs and markers, or the gene effects were not large enough to be detected in an experiment of the size conducted. It is concluded that prospects appear good for detecting QTLs and using marker-assisted selection in a tetraploid potato breeding programme, provided that, in future, the population size is increased to over 250 and more SSR markers are used to complement the AFLPs; the same is likely to be true for other autotetraploid crops.

Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.

Mitochondrial DNA differences distinguishing Meloidogyne mayaguensis from the major species of tropical root-knot nematodes

- The mitochondrial DNA region between the COII and lRNA genes and the 63 base pair tandem repeat region have been used to differentiate and characterise Meloidogyne spp. In this study these regions have been amplified from M. mayaguensis, M. javanica, M. arenaria, M. incognita and M. hapla. Meloidogyne mayaguensis produces a unique product of 705 bp from the COII and lRNA region. Also, a product of 322 bp was produced from the 63 bp repeat region of M. mayaguensis unlike M. javanica, M. arenaria, and M. incognita that exhibit hypervariability in this region. Meloidogyne mayaguensis is a widely distributed root-knot nematode with the potential to cause great economic damage. These molecular diagnostics can be used for accurate identification of M. mayaguensis and can be used to monitor the occurrence and spread of this species, and to provide quarantine services tools to limit its dispersal.

Ribosomal intergenic spacer: a polymerase chain reaction diagnostic for Meloidogyne chitwoodi, M. fallax, and M. hapla.

ABSTRACT Polymerase chain reaction amplification of the intergenic spacer region between the 5S and 18S genes from Meloidogyne chitwoodi, M. fallax, and M. hapla enabled these three important temperate species to be differentiated. Length polymorphism was found between M. chitwoodi and M. fallax as a result of differing numbers of short repeats located between the 5S and 18S genes. The presence of the 5S gene within the rDNA cistrons was confirmed in the Meloidogyne spp. included in this study. The region between the 28S and 5S genes for M. chitwoodi and M. fallax was short and lacked variability in repeated sequences compared with the main tropical Meloidogyne spp. and M. hapla. Differences in the number of these repeats resulted in intraspecific length polymorphism for M.hapla.

Variation in root-knot nematode (Meloidogyne spp.) in Crete in relation to control with resistant tomato and pepper

A targeted survey of vegetable production areas in Crete, Greece, showed that the majority of infestations of rootknot nematodes (RKN) were Meloidogyne javanica. M. incognita appeared to be limited to areas growing pepper that is a host for M. incognita but not for M. javanica. Tests with resistant tomato carrying the Mi gene showed that the M. incognita and the majority of the M. javanica populations were avirulent, and that virulent nematodes could not be selected from them. However, three populations of M. javanica, including some previously identified, were virulent. An AFLP study of the DNA from 22 populations of M. javanica produced 264 scorable amplification products and gave a very high mean similarity (99.4%) between populations, indicating that both virulent and avirulent populations probably derived from the same founder population. Mi-resistant tomato and pepper appear to have considerable utility in the integrated management of RKN in Crete, provided care is taken to monitor the species and virulence or reproductive ability of the nematode populations. Die Variation bei Wurzelgallennematoden (Meloidogyne spp.) auf Kreta in Bezug auf ihre Bekampfung mit resistenten Tomaten und Paprika - Eine gezielte Untersuchung von Gemuseanbaugebieten auf Kreta Griechenland, ergab, dass die Mehrzahl der von Wurzelgallennematoden (RKN) befallenen Flachen mit Meloidogyne javanica verseucht war. M. incognita was offentsichtlich auf Gebiete des Paprikaanbaus beschrankt. Paprika ist ein Wirt fur M. incognita aber nicht fur M. javanica . Versuche mit resistenten Tomaten, die das Mi-Gen besassen, zeigten, dass M. incognita und die Mehrzahl der Populationen von M. javanica avirulent waren, und dass aus ihnen keine virulenten Nematoden selektiert werden konnten. Drei Populationen von M. javanica, darunter schon einige schon vorher identifizierte, waren virulent. Eine AFLP-Untersuchung der DNA von 22 Populationen von M. javanica ergab 264 unterscheidbare Amplifikationsprodukte und zeigte eine hohe mittlere Ahnlichkeit (99,4%) zwischen den Populationen. Dies deutete daraufhin, das virulente and avirulente Populationen wahrscheinlich von der gleichen Grunderpopulation abstammten. Mi-resistente Tomaten und Paprika sind offensichtlich von betrachtlichen Nutzen bei der integrierten Bekampfung von RKN auf Kreta. Voraussetzung ist, dass die Arten und die Virulenz oder die Vermehrungsfahigkeit der Populationen sorgfalting festgestell werden.

The Mitochondrial Subgenomes of the Nematode Globodera pallida Are Mosaics: Evidence of Recombination in an Animal Mitochondrial Genome

We sequenced four mitochondrial subgenomes from the potato cyst nematode Globodera pallida, previously characterized as one of the few animals to have a multipartite mitochondrial genome. The sequence data indicate that three of these subgenomic mitochondrial circles are mosaics, comprising long, multigenic fragments derived from fragments of the other circles. This pattern is consistent with the operation of intermitochondrial recombination, a process generally considered absent in animal mitochondria. We also report that many of the duplicated genes contain deleterious mutations, ones likely to render the gene nonfunctional; gene conversion does not appear to be homogenizing the different gene copies. The proposed nonfunctional copies are clustered on particular circles, whereas copies that are likely to code functional gene products are clustered on others.

Parasitism genes and host range disparities in biotrophic nematodes: the conundrum of polyphagy versus specialisation

This essay considers biotrophic cyst and root‐knot nematodes in relation to their biology, host–parasite interactions and molecular genetics. These nematodes have to face the biological consequences of the physical constraints imposed by the soil environment in which they live while their hosts inhabit both above and below ground environments. The two groups of nematodes appear to have adopted radically different solutions to these problems with the result that one group is a host specialist and reproduces sexually while the other has an enormous host range and reproduces by mitotic parthenogenesis. We consider what is known about the modes of parasitism used by these nematodes and how it relates to their host range, including the surprising finding that parasitism genes in both nematode groups have been recruited from bacteria. The nuclear and mitochondrial genomes of these two nematode groups are very different and we consider how these findings relate to the biology of the organisms. BioEssays 30:249–259, 2008.

The role of flavonoids produced in response to cyst nematode infection of Arabidopsis thaliana

Flavonoids have diverse roles in plants, including defence against plant pathogens and regulation of local auxin transport. Flavonoids have been shown to be produced in feeding sites of root-knot nematodes induced in a leguminous plant, and it has previously been suggested that they may be responsible for manipulation of local auxin levels that underlie early feeding site development. Here we show that flavonoids are also produced in developing syncytia induced by Heterodera schachtii and in galls induced by Xiphinema diversicaudatum in a non-leguminous plant, Arabidopsis thaliana. We further investigated whether flavonoids are required for normal feeding site development by screening mutant lines of A. thaliana, defective in various parts of the flavonoid biosynthetic pathway, with H. schachtii. None of the lines showed a reduced capacity to support nematode infection and some showed a statistically significant increase in the numbers of female nematodes that developed. These data suggest that flavonoids are produced as part of the defence response to nematode infection rather than being an integral component of the mechanisms used by nematodes to induce feeding sites.