1. Gene expression in white clover with and without colonisation by arbuscular mycorrhizal fungi
R. Kelly1, P. E. Linton2*, W.R. Eason1, J.E Hooker2*. K.J Webb1
Institute of Grassland and Environmental Research, Aberystwyth, UK1;
Manchester Metropolitan University, Manchester, UK2; *present at conference
Table 1: Selection of sequences from leaves and root s with homology to known genes or ESTs
Introduction
White clover (Trifolium repens) is an important component of low input pasture systems and can form effective associations with both N-fixing bacteria and arbuscular mycorrhizal fungi (AMF).
Optimal functioning of legume symbiosis is central to successful development of economically and environmentally efficient agricultural forage crop systems. Symbiotic associations of legumes, such as white clover with rhizobium and AMF, provide environmental and nutritional benefits.
This study has exploited near-isogenic lines (NILs) of white clover to identify genes involved in legume symbiont interactions. These NILs are ideal for genetic studies since important agronomic traits are fixed (Eason et al., 2001) with a view to assigning functions to genes involved in these interactions and generating molecular markers for improved symbiosis in new varieties.
Conclusions
These results have highlighted differences in gene expression in mycorrhizal and non-mycorrhizal NILs of white clover. For example a gene that shares homology with the photosynthesis related gene protochlorophyllide oxido reductase is expressed in leaf tissue in plants with mycorrhizal colonisation and not in control plants. Rates of photosynthesis have been shown to be affected by AMF colonisation (Wright et al., 1998).
Rapid amplification of cDNA ends (RACE) is currently being applied to confirm and extend the obtained sequences to further elucidate the genetic factors that control AMF symbiosis in white clover.
Understanding the genetic control of plant-AMF interactions will allow clover breeders to develop genetic markers for selection of genotypes favourable to AMF symbiosis.
Methods
Differential display methods were used to identify changes in gene expression in both leaves and roots from NIL plants grown in a nutrient flowing culture system. These plants were either colonised with G. mosseae and G. intraradices and or left as control plants without colonisation with AMF.
RNA was isolated from plant tissue, reverse transcribed, amplified and visualised on PAGE gels. The mRNA was selected using polyT primers and split up into a range of different size fragments using randomly generated arbitrary primers. Bands that showed different expression patterns were excised, purified, cloned into a vector and sequenced. These sequences were compared with those in Genbank and homology noted.
Future Work
There are few reports of the identification of root senescence associated genes in clover associated with AMF colonisation and it is therefore of wide importance to characterise gene expression during root senescence in such plants to determine genetic markers which will enable selection of desirable traits.
We are now focusing on how AMF modify the growth and senescence of roots and are comparing gene expression patterns in individual roots tips with and without colonisation with G. intraradices using cDNA AFLP methods.
Results
Differential display demonstrated differences in gene expression in both leaves and roots of clover with and without AMF. These genes were mainly down regulated in AMF plants (e.g. Fig 1).
In total 45 sequences were identified and cloned; 30 from leaves and 15 from roots. Sixteen sequences had homology with known genes (for a summary (see Table 1). The majority corresponded to ESTs from model legumes and from studies in plants with phosphate and nitrogen starvation.
Some sequences showed no homology with those in the databases and so may represent previously undiscovered genes. The results obtained indicate clear differences in gene expression in white clover plants colonised by AMF
Control G. m G. i
We are using microcosms (Figure 2) that enable us to follow the growth of individual roots and allow us to observe individual root tips throughout their life cycle and extract RNA at different stages of development.
Figure 2. Defoliated red clover (Trifolium pratense) plant in a microcosm with removable front panel to enable the selection of roots of different ages and different stages of senescence. The insert shows a close-up of a section of the root system)
References
Eason, W.R., Webb, K.J., Michaelson-Yeates, T.P.T., Abberton, M.T., Griffith, G.W., Culshaw, C.M., Hooker, J.E. and Dhanoa, M.S. (2001) Effect of genotype of Trifolium repens on mycorrhizal symbiosis with Glomus mosseae. Journal of Agricultural Science 137,
Wright, D.P., Scholes, J.D. & Read, D.J. (1998) Effects of VA mycorrhizal colonization on photosynthesis and biomass production of Trifolium repens L. Plant, Cell & Environment 21,
Figure 1. Gene expression in Trifolium
repens with and without mycorrhizal
colonisation. G. m - G. mosseae and
G.i - G. intraradices.
IGER is grant aided by BBSRC