Offered Poster Abstracts - VI
Identification and characterisation of two metallothionein-encoding genes
from the rice blast fungus, Magnaporthe grisea
Magnaporthe grisea is the causal agent of rice blast disease. Considerable research has led to our current understanding of this pathogen however little emphasis has been placed on identifying genes specifically involved in plant tissue colonisation and growth of the fungus in planta. I this project we have adopted tow strategies to study plant tissue colonisation by M. grisea. The first strategy involved differential cDNA screening to isolate transcripts expressed in the wild-type strain of M. grisea, Guy-11 but not in a non-pathogenic MAP kinase mutant Dpmk1. Secondly, a candidate gene approach was used to identify a homologus of a gene identified in Uromyces fabae called PIG11. Because characterisation of PIG11 in this obligate biotrophic fungus is difficult, the presence of a homologue in the more experimentally amenable fungus M. grisea is significant. Using these approaches two metallothionein (MT)-encoding genes have been identified called MMT1 (the PIG11 homologue) and MMT2. Metaolothioneins are ubiquitous proteins with metal-binding propteries, although their function is somewhat elusive. Preliminary characterisation of the two MTs carried out to date will be presented, revealing their relatedness to other MTs, their patterns of expression in developmental mutants of M. grisea and the possible functions they may carry out during growth of the fungus in planta.
Rhizoctonia: Cerberus in the paddy fields: A new perspective on
rice sheath diseases and their causal organisms
Rhizoctonia sheath blight is a serious problem for rice growers around the world, particularly in areas of intensifying rice production. The causal organism has been identified as R. solani. However, two other species, R. oryzae and R. oryzae-sativae (causing sheath spot and aggregated sheath spot of rice respectively), produce symptoms that are often indistinguishable under field conditions, making accurate diagnosis of the particular culprit(s) in a given situation difficult. Reliance on traditional isolation-based methods is slow, subject to problems of preferential isolation, and requires the services of an expert mycologist. We report here on the successful application in India of a newly-developed PCR-based diagnostic method that can provide an accurate species diagnosis within 36 hours of receipt of infected plant samples in the laboratory. Application of this tool has revealed a greater complexity in the Rhizoctonia sheath disease problem than previously recognised. The implications of our finding for the development of appropriate disease control methods for use under sustainable agricultural systems are explored.
Detection and diversity of Fusarium solani f.sp. phaseoli from
common beans in south-western Uganda
Fusarium solani f.sp. phaseoli is one of a complex of organisms that can cause bean root rot disease, currently epidemic in the Great Lakes Region of Africa. Molecular studies have been initiated with the aims of (i) development of specific detection systems for this pathogen and (ii) examining population diversity. RFLPs of PCR products of the ITS region have successfully differentiated F. oxysporum and F. solani isolates from bean plants showing symptoms of root rot. A pair of PCR primers designed from the ITS region of Fusarium solani f.sp. phaseoli amplified target DNA from F. solani but not from several other Fusarium species. Molecular variation within a set of F. solani isolates collected from bean plants in south-western Uganda has been evaluated using RAPDs. The pathogenicity of these isolates is being determined and, in conjunction with RAPD data, may allow us to identify pathogen-specific markers. These, in turn may permit the design and utilisation of pathogen-specific PCR primers.
Basis of differences in aggressiveness between Microdochium nivale
isolates on rye grass
Microdochium nivale causes pink snow mould on winter cereals and grasses in the Nordic countries. At warmer temperatures it also causes leaf blotch, stem rot and head blight. Based on morphological traits, the species has been divided into two varieties, var. nivale and var. majus. We have isolated more than 30 M. nivale strains from different grass and cereal hosts. Individual isolates displayed variation in aggressiveness on rye grass, as measured by a plant re-growth assay after infection and incubation under artificial snow cover. Selected isolates were also inoculated onto winter wheat and other grass and cereal species. These isolates showed variation in aggressiveness on all hosts examined. The relative aggressiveness of individual isolates was not the same on all host plants, suggesting that factors in both plant and pathogen are involved in pathogenicity.
To better understand the basis for differences in aggressiveness on a single host plant, rye grass, we examined various characteristics of a selection of M. nivale isolates under culture conditions. Rates of growth on a rich culture medium (PDA) were measured at several different temperatures. Growth rates at 2C (the temperature under artificial snow cover) were generally low for M. nivale var. majus isolates, but were variable for M. nivale var. nivale isolates. There was no strict correlation between growth rates in culture and aggressiveness on rye grass (or on wheat), suggesting that factors in addition to robust growth at (low) temperature are involved in pathogenicity.
To determine what factors in addition to growth rate might contribute to differences in aggressiveness on rye grass, we examined the profile of proteins secreted by different M. nivale isolates into a basal salts medium containing rye grass cell walls as the carbon source. There were differences in the timing and specific activity of cell wall-degrading enzymes secreted into the medium by individual isolates. These differences could account for some of the additional variation in aggressiveness observed between M. nivale isolates.
Geminiviral AC2, a pathogenicity determinant
AC2 (also known as AL2 or C2), encoded by the members of Begomoviruses of the Geminiviridae, is a transcriptional activator protein. It transactivates viral coat protein and movement protein gene expression. Recent work on the African cassava mosaic virus (ACMV) has indicated that direct expression of AC2 protein induces necrosis in plants, implying its role in pathogenicity, and AC2 protein acts as a suppressor of post-transcriptional gene silencing. Here, we present that AC2 of the Tomato yellow leaf curl virus (TYLCV), localising in the nucleus, is a determinant of viral pathogenicity. To express TYLCV AC2 in Nicotiana benthamiana, the coding sequence for wild-type AC2, its mutant derivatives, and AC2-GFP fusion protein were cloned into a potato virus X (PVX)-based vector. These PVX RNA transcripts produced by in vitro transcription were infectious to N. benthamiana. Expression of wild-type TYLCV AC2 and AC2-GFP fusion protein induced necrotic ringspots on the inoculated leaves, rather than necrotic lesions induced by the ACMV AC2. The nucleus localisation of GFP fluorescence in the plant cells infected with PVX/AC2-GFP clearly indicated AC2 translocated GFP into the nucleus, which was further evidenced by that AC2-mediated translocalisation of GFP into insect cell nucleus.
Influence of Pseudomonas fluorescens strain PSR 21 on the
alkylresorcinols composition in barley and their potential antifungal activity
Pseudomonas fluorescens strain PsR 21 previously isolated from the rhizosphere of cannola (Brassica napus ssp. oleifera L.) had shown the ability for plant growth promotion of some field cultivated crops. We studied the influence of seed treatment with the strain on the yield, the seed infestation as well as on the quantity and quality of naturally occurring non-isoprenoid phenolic lipids, 5-alkylresorcinols in seeds after harvest under field conditions. These polyketide-derived, odd-numbered, long-chain homologues of orcinol (1,3-dihydroxy-5-methylbenzene) are constitutively present in barley both in all vegetative organs and grains. Due to their strong antifungal activity and their localisation in the hydrophobic epicuticular wax layer, alkylresorcinols are an important protective factor in biology of barley grains and seedlings against external aggression and predators.
For the purpose of this research, seeds of barley cv. Rudzik were inoculated before sowing with a suspension of P. fluorescens PsR 21 cells. Control seeds remained untreated. The effect of the seeds treatment resulted in significant increase of the yield in comparison with control plants. Moreover, we clearly observed a lower number of colony forming units of Atlernaria alternata, Botritis cinerea and general number of fungi on seeds harvested from plots treated with P. fluorescens PsR21 than from control ones.
Both control and inoculated plants contained comparable amounts of alkylresorcinols. However, some differences in homologue compositions were being observed. Plants treated with the bacterium as well as control plants biosynthesised the same homologues with carbon side-chains from C17 to C25. In comparison with control, the relative content of the short-chain alkylresorcinols (C17 and C19) in tested plants was decreased, and that of the longest homologue (C25) increased. Recently, we have proved that antifungal activity of saturated alkylresorcinols is in direct proportion to the carbon side-chain length. Thereby, induction of long-chain alkylresorcinols in barley improves the activity of such mixtures against some undesirable phytopathogens. Thus, direct interactions between barley and the tested pseudomonad resulted in the induced plant-host resistance acquisition versus certain phytopathogenic microbes.
Undoubtedly, this finding has proved that changes in alkylresorcinol homologue ratios in barley grains may be one of still weakly recognised mechanisms of actions of P. fluorescens strain PsR 21.
Extracellular matrix and surface attachment of Stagonospora nodorum sporelings:
an immunocytochemical analysis
Stagonospora nodorum (Berk.), previously known as Septoria nodorum (Berk.) Berk. is an air borne foliar pathogen of cereals. It is responsible for leaf and glume-blotch disease and is common in temperate climates such as the UK. S. nodorum often appears in the field together with the fungal leaf pathogen, Septoria tritici1. Together they are known as the Septoria spp. complex, and account for a worldwide annual yield crop loss estimated at around 0.6 billion.
Most of our current knowledge focuses mainly on the molecular genetics of S. nodorum and in studies of the advanced stages of the disease. However, it is now recognised that new strategies in the development of early disease control lay in close inspection and detailed understanding of the initial stages of infection. Thus, the initial stages of fungal contact with the host surface play a crucial role in subsequent infection2.
The aim of this study was to investigate these early stages focusing on the production of the fungal extracellular matrices (ECMs) and the adhesion of sporelings to the wheat leaf surface. Understanding such mechanisms could provide a new insight into the control of leaf and glume-blotch.
Monoclonal antibodies (MAbs) SN.MG11-EF7, SN.CH9-EG8 were raised against S. nodorum surface molecules and recognise a protein epitope on the surface of fungal walls or a carbohydrate epitope present in the secreted extracellular matrix. The possible roles of these S. nodorum antigens in the early host-pathogen interaction were studied. A variety of light and electron microscopy methods were employed to visualise the fluorescent or gold-labelled MAbs and were used to document the spatial relationship between sporeling attachment, ECM secretion and the host surface.
The results suggest that there is a rapid and strong adhesion of the ungerminated conidia to the leaf surface. The ECM appears to be secreted in two stage-specific phases, notably from the ungerminated pycnidiospores and around emerged germ-tubes. The level of secretion from pycnidiospores appears to be dependent on substratum surface factors and upon relative humidity.
1. Shaner G. and Buechley G. 1995. Epidemiology of Leaf Blotch of Soft Red Winter Wheat Caused by Septoria tritici and Stagonospora nodorum. 79:928-938
2. Nicholson R.L., Epstein L. 1991. Adhesion of fungi to the plant surface. In: The fungal spore and Disease initiation in plant and animals (ed. G.T Cole and H.C Hoch) New York , Plenum Press. pp: 3-23
Isolation of genes induced during compatible interactions between leaf
rust (Puccinia recondita) and wheat
The rust fungi are obligate biotrophic pathogens that depend on living host tissue for their growth. In compatible interactions they go through a number of development stages to form intercellular hyphae and haustoria within host cells, through which they obtain nutrients to support colony growth. Therefore, the isolation of genes induced in both host and pathogen during their compatible interaction may provide an approach for understanding the molecular mechanism of disease development in the biotrophic plant pathogens. Here we have exploited the cDNA-AFLP technique to isolate wheat and wheat leaf rust genes expressed at specific defined time-points during the infection process. Most of genes isolated from the cDNA-AFLP showed the identical expression patterns as Northern blotting analysis. Sequence analysis has revealed similarities amongst the fungal genes to bacterial, fungal and yeast chitinase, sorbitol dehydrogenase, proteaseome regulatory unit and tyrosinase, whilst in wheat, we have identified sequences with homology to Arabidopsis katanin and cell enlargement protein. The origin of wheat or rust genes has been conformed by PCR and Southern analysis. A cDNA library constructed from post-inoculation pooled cDNAs from wheat leaves has been screened to isolate the full-length cDNAs for further analysis.
Walking into the unknown: a "step down" PCR-based technique
leading to the direct sequence analysis of flanking genomic DNA
We describe a novel and efficient PCR-based technique for walking into unknown flanking genomic DNA without recourse to protracted laborious library screening for overlapping sequences. This two component "hot start" and "step down" PCR method uses 6 x 1m g of genomic DNA (c. 20 kb in length) restricted with 6 different endonucleases and ligated to adaptors with the inclusion of 2 further restriction enzymes to prevent self-ligation. It allowed us to walk, in a single step, up to 6 kb into flanking Erysiphe graminis DNA and gave sufficient PCR products for up to 200 different walking experiments. This technology enabled us to clone and characterise the previously elusive 5 sequence of the barley powdery mildew chitin synthase gene, BgChs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene1,2
To-date, using this technique, we have gathered rapidly genomic sequence data from a further 16 mildew genes and 10 promoter sequences. We have performed more than 60 walking steps, yielding some 60 kb of sequence in rapid succession and without a single failed attempt.
1. Zhang, Z., Gurr, S. J. (2000). Walking into the unknown: a 'step down'
PCR-based technique leading to the direct sequence analysis of flanking genomic
DNA. Gene 253 (2), 145-150.
A pharmacological and molecular approach to the study of signal
transduction in the barley powdery mildew fungus
Blumeria graminis is the causal agent of barley powdery mildew disease. Infection is spread by asexual conidia, which, on contact with the leaf surface, undergo a complex and highly regulated programme of development. Conidia germinate and produce a short primary germ tube followed by a second formed germ tube which elongates, swells and produces a specialised infection structure, the appressorium.
B. graminis is an obligate biotroph, meaning that it cannot be grown axenically and consequently, tissue for experiments is limiting. Thus, we have employed and described a range of techniques to assess how B. graminis perceives, integrates and relays signals for morphogenesis up to the point of penetration. Previous work has demonstrated that both physical properties of the leaf surface, such as hydrophobicity, and cuticle-derived chemicals, such as cutin monomers and cellulose, promote B. graminis differentiation. But how does B. graminis transduce signals to drive differentiation and development? Applications of exogenous agonists and antagonists have allowed us to demonstrate a role for cAMP signalling and PKA in germling differentiation, but this work also highlights that cAMP alone is not sufficient to trigger the complete programme of differentiation.
We have identified several component genes of signal transduction and cell integrity pathways in B. graminis, including two PKC genes, two MAPK genes and two chitin synthase genes. Their expression profiles show that they are regulated differentially during conidia germination and appressorial differentiation. They putatively play important roles in host penetration and pathogenicity. We aim to ascribe functions to these genes, by using our recently-described stable DNA transformation technique and also to study the interplay between the PKA, PKC and MAPK signal transduction pathways.
PR-10 genes of the apple seedlings : analysis of regulation and
spatio-temporal expression after induction by acibenzolar-S-methyl (an analogue
of salicylic acid)
A large number of acidic PR proteins presenting similar molecular weight (16 to 19 kDa) and amino acid sequences have been grouped into the PR-10 family (Van Loon et al., 1994). Their intracellular localisation and the presence of pollen and food allergens in this family (Breiteneder et al., 1989; Vieths and Schning, 1996) constitute two specific features of this family of defense proteins. PR-10 proteins, have been identified in various organs in numerous plant species but little is know about their expression patterns and genes regulation.
Four PR-10 transcripts named AP2, AP3, AP4 and AP5 have been identified in the young leaves of apple seedling (Malus domestica Golden Delicious) after treatment with acibenzolar-S-methyl (ASM, a synthetic analogue of salicylic acid). These transcripts have been grouped on the basis of their amino acid sequences in two subfamilies: APa, grouping AP2 and AP5 and APb grouping AP3 and AP4. The expression of the APa and APb genes were analysed between 8 h and 48 h after treatment with ASM at the transcripts level by RT-PCR and northern blot, and at the protein level by western blot. Results showed that these two subfamilies are induced by ASM with a strong accumulation of transcripts between 20 h and 48 h. Otherwise, the immunoblotting (using antibodies raised against the major birch pollen allergen Bet v1) revealed the presence of two bands of 17 and 18 kDa at 48 h after treatment by ASM. Northern blot analysis of PR-10 genes expression also showed that there was a systemic accumulation of transcripts of the two subfamilies APa and APb at 120 h after application of ASM. The immunolocalisation of PR-10 proteins in the young leaves of apple seedlings showed that they are mainly localised in vascular tissues. Promoters of the four genes (AP2, AP3, AP4 and AP5) have been cloned with the aim of analysing their nucleotide sequences in order to identify the potential fixation sites for transcription factors. Subsequently, constructions with reporter genes will be achieved to analyse the in vivo regulation of the expression of these genes.
Breiteneder H., Pettenburger K., Bito A., Valenta R., Kraft D., Rumpold H.,
Scheiner O. and Breitenbach M. 1989. The EMBO J. 7, 1935-38.