Offered Poster Abstracts - IV
Virus-induced gene silencing in a high throughput system to identify genes
involved in Rx- and pto-mediated resistance
Virus induced gene silencing (VIGS) is a process whereby virus infection causes sequence specific down-regulation of plant RNA(s) sharing homology with the infecting virus.
We have developed a high throughput system for VIGS of random genes. A Nicotiana benthamiana cDNA library was normalised and cloned into a binary PVX vector. The PVX-cDNA library was then transformed into Agrobacterium and a direct inoculation technique for PVX infection was developed.
A random screen was carried out to identify genes required for Rx- and Pto- mediated disease resistance. Resistance assays were performed on optimally silenced leaves. These assays are dependent on the hypersensitive response (HR) which arises after infiltration of Agrobacterium expressing the resistance gene and its corresponding Avr product. If a gene required for resistance ( for example Prf in the case of Pto resistance) is silenced, we do not observe a HR.
Of approximately 5000 genes, we 62 candidates required for the Pto HR, 12 candidates for the Rx HR and 17 candidates for both resistances. We are now further testing these candidate genes by challenging plants transgenic for Pto or Rx with the corresponding pathogens. Thus PVX-GFP or P. syringae (carrying avrPto) are used to assay loss of Rx-mediated or Pto-mediated resistance respectively. To date, candidate genes in the Pto reistance pathway have been confirmed using the bioassay. Further analysis is being carried out to investigate the structure and function of these candidates.
Baulcombe D.C. 1999.Fast forward genetics based on virus-induced gene silencing. Current Opinion In Plant Biology.2,109-113.
Cloning and characterisation of avirulence gene Avr2 of Cladosporium
The interaction between tomato and the biotrophic fungus Cladosporium fulvum complies with the gene-for-gene model. The tomato resistance locus Cf-2 contains two homologous genes, Cf-2.1 and Cf-2.2. Both genes confer a hypersensitive response (HR)-mediated resistance to isolates of C. fulvum producing the matching elicitor. Attempts to clone the Avr2 gene by reverse genetics have not been successful. Therefore, a PVX-based binary expression vector was used to allow Agrobacterium tumefaciens-delivered functional expression of a cDNA library of C. fulvum in tomato plants (Takken et al., Plant J., in press).
Upon toothpick inoculation of Agrobacterium colonies onto tomato leaves, five independent clones, containing an identical open reading frame (ORF), were identified that gave Cf-2-specific HR. Avr2 encodes a cysteine-rich protein of 78 amino acids (AA), with a predicted signal peptide for extracellular targeting of 20 AA. Tobacco lines expressing either Cf-2.1 or Cf-2.2 responded with a HR upon AVR2, indicating that both Cf-2 genes confer AVR2 recognition. Strains of C. fulvum virulent on Cf2 tomato plants circumvent recognition by various single mutations in the ORF of the Avr2 gene, that either result in a frameshift or in the insertion of a stopcodon. To prove that Avr2 is indeed responsible for avirulence of C. fulvum on Cf2 plants, a strain virulent on Cf2 plants will be transformed with the Avr2 gene.
Compatible powdery mildew infection suppress the hypersensitive response
to incompatible mildew in Mla-1 resistant barley.
Two different Mla-1 resistant barley lines were sequentially inoculated with compatible (virulent) and incompatible (avirulent) powdery mildew (Blumeria graminis f.sp. hordei) isolates. When the barley lines were attacked by the compatible B. graminis isolate, infection attempts either failed due to papilla formation or succeeded and a haustorium was formed in the attacked epidermis cell. However, when leaves were attacked by the avirulent B. graminis isolate, a very high percentage of attacked epidermal cells die in a rapid, hypersensitive response which is almost always restricted to the single cell under attack
The double inoculation treatments were performed, by applying first the compatible inoculum, then incubate for 48 h before removing superficial fungal structures leaving epidermal cells that either contained an inducer haustorium or a papilla. The second, incompatible challenge inoculum was then applied and incubated for 48 h before preparation for histological analysis (Lyngkjr & Carver 1999a ).
As previously demonstrated where a papilla due to inducer attack was present, the cell, and to some extent its neighbours, showed induced inaccessibility (preventing fungal penetration by papilla response) (Lyngkjr & Carver 1999a & b, Carver et al., 1999). By contrast, when compatible inducer haustoria formed in Mla1 epidermal cells, these cells not only showed very high accessibility to challenge attack by the incompatible isolate, but also the plant cell survived in the presence of the incompatible haustorium, and vigorous colonies developed. Despite this, mesophyll underlying such epidermal cells sometimes showed extensive whole-cell autofluorescence indicating that they were dying or dead. It appears that establishment of the compatible inducer infection maintained vitality of the epidermal cell containing the incompatible haustorium but that signals from the incompatible fungus disrupted the underlying mesophyll generating a novel response phenotype.
Carver TLW, Lyngkjr MF, Neyron L, Strudwicke CC (1999): Induction of cellular accessibility and inaccessibility and suppression and potentiation of cell death in oat attacked by Blumeria graminis f.sp. avenae. Physiological and Molecular Plant Pathology 55:183-196
Lyngkjr MF, Carver TLW (1999a): Induced accessibility and inaccessibility in barley epidermal cells by a compatible Blumeria graminis f.sp. hordei isolate. Physiological and Molecular Plant Pathology 55:151-162.
Lyngkjr MF, Carver TLW (1999b): Modification of mlo5 resistance to Blumeria graminis attack in barley as a consequence of induced accessibility and inaccessibility. Physiological and Molecular Plant Pathology 55:163-174.
Morphological and molecular identification of Pythium species
pathogenic to common beans in Uganda
The common bean (Phaseolus vulgaris L.) is one of the most important sources of dietary protein and calories produced in Uganda. Bean yield is estimated at 349 kg/ha compared to 787 kg/ha ten years ago. The main causes of this reduction in bean yield are declining soil fertility and the effect of insect pests and diseases, most specifically root rots. One of the major pathogen genera causing severe bean root rots in Uganda has been identified as Pythium, other genera involved are Fusarium and Rhizoctonia. The identification of Pythium to species level in this disease complex is critical for effective epidemiological studies leading to control strategies. The aim of this study is to identify bean pathogenic strains of Pythium spp. using both morphology and DNA-based molecular markers. Samples of bean plants displaying root rot symptoms were collected from three regions in Uganda. Twenty-one Pythium strains identified to species or groups using morphology were considered for the preliminary study. These Ugandan strains were compared with selected Pythium species obtained from culture collections. The ITS spacer regions flanking the 5.8S rRNA gene were amplified and digested with one of the following restriction enzymes: CfoI, MboI, HinfI and TaqI. Groupings arising from RFLP banding pattern were compared with groupings based on morphology. This work will be linked with results from pathogenicity studies currently in progress using representative isolates from each group, which may be useful in identifying pathogen specific markers.
Natural variation in Arabidopsis reveals multi-component resistance
to the downy mildew Peronospora parasitica(At) isolate Cala2
Resistance to Peronospora parasitica (At) isolate Cala2 in Arabidopsis accession Col-5 has been mapped to a single locus, RPP2, on chromosome 4. A gene, RPP2A, has been cloned and shown to complement a Cala2 susceptible mutant. However, we have shown that a second gene required for resistance to Cala2 is closely linked to RPP2A. To identify this second component susceptible F2s from the Col-5 x Nd-1 cross are being screened for individuals that contain RPP2A. These individuals will contain recombination events between RPP2A and the second component, allowing the mapping interval of the second resistance gene to be reduced to a very small region. A second approach to identify the gene is to sequence candidate genes closely linked to RPP2A from mutants shown to be altered in the function of the second component of Cala2 resistance. Progress towards cloning the second component will be reported.
Transient expression of the green fluorescent protein in Nicotiana
benthamiana mimics pathogen attack?
The jellyfish green fluorescent protein (GFP) is probably the most popular reporter gene currently in use. It has been used extensively as a "tag" to monitor the cellular localisation and intra- and intercellular movement of proteins, organelles, and viruses and as a reporter gene in transgenic plants.
In this study we investigated whether expression of GFP in Nicotiana benthamiana alters plant gene expression. We introduced a 35S-GFP5 construct into plants using a transient expression assay based on Agrobacterium tumefaciens infiltration of leaves. The mRNAs produced in these leaves were compared with those produced in leaves infiltrated with control constructs (35S-GUS, 35S-GFP4, and vector alone constructs) using cDNA-AFLP analyses. 53 AFLP bands were induced and 10 AFLP bands were repressed specifically by 35S-GFP5. Database searches revealed that most of the induced bands showed homology with proteins involved in plant defence.
The genes were not induced in 35S-GFP5 transgenic N. benthamiana plants but could be induced by Agrobacterium infiltration of the 35S-GFP5 construct. As in the non-transformed plants, none of the control infiltrations induced gene expression.
The GFP5 sequence differs from GFP4 in that it has a chitinase (CHT) signal peptide at the N-terminus (which targets the protein to the endoplasmic reticulum (ER)) and an ER retention signal at the C-terminus. To determine whether the CHT signal peptide or localisation of foreign proteins to the ER induces gene expression, we also infiltrated a 35S-CHT-GUS construct and a 35S-VIC-GFP construct (VIC; the vicilin storage protein signal peptide). The data from these cDNA-AFLP analyses will also be presented.
Identification of genes required for N-mediated resistance against TMV by
virus-induced gene silencing
The aim of this project was to identify genes required for the N-mediated defence response against tobacco mosaic virus (TMV).
Infection of plants by a virus carrying a fragment of a host gene leads to suppression of the corresponding host gene in a process termed virus induced gene silencing (VIGS). Here VIGS was exploited to identify genes required for N-mediated resistance; silencing genes necessary for N function will break resistance and enable TMV susceptibility.
Nicotiana benthamiana plants are amenable to VIGS. Thus an N genomic fragment from tobacco was used to transform N. benthamiana plants. N transgenic plants were resistant to recombinant TMV isolates demonstrating that components necessary for N function are likely to be conserved between tobacco and N. benthamiana.
In order to validate the notion that VIGS could be used as a tool to identify components of the N resistance response, N itself was targeted for suppression. Infection of N transgenic plants with virus vectors carrying a fragment of N led to silencing of N and TMV susceptibility.
The requirement of EDS1 in the N resistance pathway was then tested. VIGS of a N. benthamiana EDS1 homologue compromised N resistance; TMV replication on EDS1 silenced plants occurred to a similar extent as on N silenced plants. These observations provide evidence that EDS1 is required for function of TIR-NBS-LRR resistance genes in species other than Arabidopsis.
Finally, VIGS was used to identify a novel N resistance pathway gene. A normalised N. benthamiana cDNA library was cloned into a potato virus X (PVX) vector. 5 000 N transgenic plants were inoculated with PVX-cDNA constructs from the library to induce silencing of corresponding genes. The plants were then screened for loss of N resistance. The N response was consistently compromised by VIGS of NRG1 (for N requirement genes). NRG1 is predicted to encode a non-TIR NBS-LRR protein. Transient over-expression of NRG1 elicited a hypersensitive response in the absence of N or the elicitor of N implying that NRG1 functions downstream of N. VIGS of NRG1 in non-transgenic N. benthamiana, i.e. TMV compatible plants, did not enable enhanced TMV replication. NRG1 silencing did not suppress the resistance response mediated by Rx or by Pto.
In summary, VIGS was used to demonstrate that EDS1 is a necessary component of the N resistance response and that N function depends on another NBS-LRR encoding gene, NRG1.
The role of alkylresorcinols in protection of cereal seedlings against
infection of some pathogenic fungi
The naturally occurring polyketide-derived phenols, 5-n-alk(en)ylresorcinols (ARs) showed significant antifungal activity in vitro versus Rhizoctonia cerealis and Rhizoctonia solani, but significantly lower against Fusarium culmorum. Due to their strong antibacterial and antifungal activity, those ARs are biosynthesised to protect the plant against pathogens. The objective of this study was to determine the sensitivity of barley, rye and wheat cultivars, which display significantly different concentrations of ARs in the waxy epicuticular layer of grains. Seeds of tested cereals were sown in sand artificially infested with Rh. cerealis or Rh. solani. Additionally, we compared the sensitivity of seedlings to fungal infection after 10-seconds washing away waxy epicuticular layer of grains with chloroform.
The infection of seedling was monitored during first five days. The seedlings of cultivars with higher amount of ARs in the waxy epicuticular layer were infected in smaller degree by tested Rhizoctonia strains. The comparison of seedlings of the same cultivar with various quantities of ARs has also proved our observations mentioned above.
We did not observed any effects of chloroform on growth and germination of seeds in control pots without pathogens. Drastically differentiations were observed in susceptibility for Rhizoctonia infections between chloroform treated and control seeds germinated in infested pots. The number of germinated seedling decreased in the range from 20% to 60% after removing the waxy epicuticular layer in comparison with control seeds in infested pots. Additionally, we observed significant decline of the length of roots (15 - 45%) as well as the increase of number and size of lesions (20 - 60%) on the roots of seedlings grown from chloroform treated seeds.
The result has confirmed the antifungal activity of analysed phenolic compounds against Rhizoctonia fungi in vitro. The ability to production of saturated resorcinols could be taken into account as one of possible factors of plant resistance against fungal infection during germination.
Biochemistry of the tomato disease resistance gene Cf-2
The tomato Cf genes confer resistance to races of the leaf mould fungus Cladosporium fulvum expressing the corresponding avirulence (Avr) genes, in a gene-for-gene manner. The Cf genes are predicted to be predominantly extracellular membrane bound glycoprotein receptors, which recognise fungal Avr gene products. One Cf gene, Cf-2 encodes 38 extracellular-type leucine rich repeats, a single potential transmembrane domain and a 37 amino acid tail, predicted to be cytoplasmically located (Dixon et al 1996). Recently studies examining the subcellular localisation of Cf-9 have produced conflicting results. Using an epitope tagged version of Cf-9 Piedras et al. (2000), demonstrated a plasma membrane localisation, whilst Benghezal et al. (2000) using both fusion proteins and epitope tagging revealed an endoplasmic reticulum localisation.
To resolve the issue of subcellular localisation, we have produced myc epitope tagged versions of Cf-2 under the control of its native promoter. These are being transformed into both tomato and tobacco. Functionally active transgenic plants carrying these constructs will be used for subcellular fractionation studies.
Benghezeal, M., Wasteneys, G.O., Jones, D.A. 2000. The C-terminal dilysine motif confers endoplasmic reticulum localization to type I membrane proetins in plants. Plant Cell. 12: (7) 1179-1201.
Dixon, M.S., Jones, D.A., Keddie, J.S., Thomas,C.M., Harrison, K., Jones, J.D.G. 1996. The tomato Cf-2 disease resitance locus comprises two functional genes encoding leucine rich repeat proteins. Cell. 84: 451-459.
Piedras, P., Rivas, S., Droge, S., Hillmer, S., Jones, J.D.G. 2000. Functional, c-myc-tagged Cf-9 resistance gene products are plasma membrane localised and glycosylated. Plant Journal. 21: (6) 529-236.
Type III Secretion in Root-Colonising Pseudomonas
Type III protein secretion is used by plant-associated bacteria to deliver proteins into the apoplast and cytoplasm of plant cells. Plant pathogens such as Pseudomonas syringae use type III secretion to promote parasitism and virulence in compatible host plants, however, type III secreted proteins can also act as elicitors of the hypersensitive response in non-host and resistant plants. We have characterised a type III secretion pathway from the plant growth-promoting bacterium Pseudomonas fluorescens SBW25 that is very similar to the type III secretion pathway of P. syringae, and have shown that type III secretion genes are widespread in both pathogenic and plant growth-promoting plant-associated Pseudomonas strains. P. fluorescens SBW25 possesses a 20 kb gene cluster which encodes both the type III secretion pathway (rsp genes) and a putative type III secreted protein RopE, similar to the type III secreted proteins AvrE and DspE from P. syringae and E. amylovora respectively. Although the organisation and sequence of the P. fluorescens cluster is very similar to the Hrp gene cluster of P. syringae, it does not contain all the components of the conserved type III secretion mechanism, or a homologue of the glycine-rich type III accessory proteins known as harpins. P. fluorescens SBW25 can elicit a type III-dependent HR in Arabidopsis thaliana and Nicotiana clevelandii leaves when the positive regulatory proteins RspR and RspL are constitutively expressed. However, rsp genes are not induced in the leaf apoplast in wild-type bacteria, which fail to elicit a visible HR on any host plant tested. Current evidence suggests that the primary location of rsp expression and activity is the rhizosphere. We have used reporter gene fusions to investigate the regulation and expression of rsp genes, and have found that although the elements of the rsp regulatory cascade function similarly to the hrp regulatory genes of P. syringae, the environmental signals that induce rsp gene expression are quite distinct. In addition, we have used rsc, rsp and ropE mutants to investigate the role of type III secretion in rhizosphere colonisation, and have found that type III secretion is not required for colonisation of sugar beet seedlings.