ICPP98 Paper Number 5.5.3S

5.5.3S
FUNCTIONAL ANALYSIS OF MGATR1 AND MGATR2, TWO ABC TRANSPORTERS OF MYCOSPHAERELLA GRAMINICOLA, THE CAUSAL AGENT OF SEPTORIA TRITICI LEAF-BLOTCH OF WHEAT
L.-H. ZWIERS and M.A. DE WAARD
Department of Phytopathology, Wageningen Agricultural University, Wageningen, The Nethedands

Background and objectives
ABC-transporters are membrane bound ATP-hydrolyzing transporters with a characteristic domain organization. These transporters occur abundantly in both pro- and eukaryotic organisms, and can have a broad substrate range.[1] A generally accepted function of ABC-transporters is the protection of organisms from poisoning by naturally cytotoxic compounds by preventing accumulation in cells. An additional effect of this function can be the development of multidrug resistance to chemotherapeutic drugs and fungicides in mammalian tumors and plant pathogens, respectively. Besides acting as a general protection mechanism against accumulation of erogenous toxins, ABC-transporters can have distinct functions like the secretion of mating factors. In plant pathogenic fungi the function of ABC-transporters can also involve (i) protection of the fungus during pathogenesis against plant defense products, (ii) secretion of pathogenicity factors (e.g. toxins), and (iii) secretion of mating factors. [2] These hypotheses are tested for the causal agent of Septoda thtici leaf-blotch of wheat,Mycosphaerelia graminicoia (anamorph Septoria tritici). Typical symptoms of diseased leaves are necrotic lesions. Formation of these lesions might be associated with the secretion of a phytotoxic pathogenicity factor by the pathogen. Wheat is also known to produce fungitoxic compounds.

Materials and methods
A genomic library from M. graminicoia

isolate IP0323 was screened with a probe derived from PDR5, a well characterized ABC-transporter gene from Saccharomyces cerevisiae. [3] Positive phages were subcloned and the clones were sequenced. This led to the cloning of tm ABC-transporter encoding genes Mgatrl and Mgatr2. Different compounds were tested for their capacity to induce expression of either of the two genes. Transformations are being performed aimed at disrupting the two genes.

Results and conclusion
The heterologous hybridization led to the isolation of only two ABC-transporter encoding genes, although probably much more are present. Currently, a PCR-based strategy has lead to the cloning of additional ABC-transporter encoding genes.[4] The cloned Mgatrl and Mgatr2 genes were sequenced and characterized. Both genes are single copy genes.
The Mgatrlgene contains 2 introns and encodes a protein of 1562 amino acids. The Mgatr2 gene contains 7 introns and encodes a protein of 1499 amino acids. Both deduced proteins contain the typical characteristics of ABC-transporters and show a high degree to other cloned fungal ABC-transporters with the [NBF-TMD6]2 configuration. Expression studies show that both genes have a different expression pattern. Mgatr1 has a low basal level of expression which increases upon treatment with cycloheximide and the plant secondary metabolise eugenol. Mgatr2 shows a more specific expression pattern. No basal expression can be detected but the gene is induced after treatment with different compounds, amongst other's the fungicide imazalil and the plant secondary metabolise eugenol. Furthermore, expression patterns seem to be different for the hw morphological states (yeast-like and mycelium) of this dimorphic fungus. These results suggest that indeed ABC-transporters play a role in pathogenesis and possibly also in resistance to certain fungicides. No transformants have yet been obtained in which one of the two genes was deleted. Thus no mutants have yet been tested for altered phenotypic traits like virulence on wheat and sensitivity to toxins.

References
1. Seelig A, 1998. European Journal of Biochemistry 251 (1998) 112, 252-261 2. De Waard MA, 1997. Pesticide Science 51, 271-275 3 Batzi E, Wang M, Leterme S, Van Dyck L, Goffeau A, 1994. Journal of Biological Chemistry 269, 2206-2214 4. Venema K, De Waard MA, 1998. Poster ICPP98

5.5.3S
SIMULATION OF THE FOLIAR UPTAKE OF SEMI-VOLATILE FUNGICIDES

M RIEDERER^l, A DAISS², N GILBERT² and H KOHLE³

^lLehrstuhl fur Botanik 11, Universität Würzburg, D-97082 Würzburg, Germany; ²BASF AG, Technische Entwicklung, D-67056 Ludwigshafen, Germany; ³BASF AG, Landwirtschaftliche Versuchsstation, D-67114 Limburgerhof, Germany

Background and objectives
Most systemic fungicides are applied to the above-ground surfaces of crops. The primary site of contact with the plant is the cuticle which represents the interface between the deposit of formulation and the internal plant tissues. Non-volatile active ingredients of the formulation can enter the plant exclusively via the cuticular pathway [1]. However, active ingredients having a finite vapour-pressure at environmental temperatures may, in addition to cuticular penetration, also be taken up through open stomata [2]. A prerequisite for this mechanism of uptake is the presence of active ingredients in the vapour phase of the boundary layer of the leaf. The objective of the present study was to investigate this mechanism of foliar uptake by a numerical simulation of the diffusion and convection processes involved.

Results and conclusions
Three model systems with increasing complexity and closeness to the real situation at the leaf surface were studied. Diffusive and convective transport in the leaflatmosphere interface was simulated using a finite elements approach. The three-dimensional model systems were translated into appropriate grid structures and numerical simulations performed. The simulations were based on the physical-chemical properties of fenpropimorphe, kresoxim-methyle and epoxiconazole which were taken as reference compounds. Diffusion coefficients of 1x10-10 M 2 S-1, 1 X, 0-11 M2 S-1, 1 X, 0-1 1 M 2 S-1 and 1x10-5 M 2 S-1 were assumed for the cell wall, cuticular polymer matrix, cuticular wax and atmosphere, respectively.
The simulations demonstrate that the rates of vapour-phase uptake of active ingredients into the interior of a leaf strongly depend on the physical-chemical properties of the chemical. Low vapour pressures and high solubilities in cuticular wax as well as high vapour pressures are unfavourable for the build-up of sufficiently high concentrations of the active ingredient in the boundary layer of the leaf. Active ingredients meeting the delicate balance between vapour pressure and wax solubility may effectively be transiocated in and taken up from the unstirred layers of air covering leaf or fruit surfaces. Active ingredients meeting the delicate balance between vapour pressure and wax solubility may effectively be transiocated into the undisturbed layers of air covering the leaf or fruit surfaces, and as a consequence a larger part of them can be taken up into the plant via the stomata.

< b> References
1. Schonherr J, Riederer M, 1989. Rev. Environ. Contam. Toxicol. 108, 1-70.
2. Riederer M, 1995. In: Plant contamination: Modeling and simulation of organic chemical processes (Trapp S, McFariane C, eds), Boca Raton: Lewis Publishers, pp. 153-190.