1.14.6
PLASMA MEMBRANE H+-ATPASE EXPRESSION DURING INFECTION WITH SEDENTARY PLANT-PARASITIC NEMATODES

N VON MENDE1, M OUFATTOLE2, M ARANGO2, M BOUTRY2 and RA LEIGH1

1IACR Rothamsted, Harpenden, Herts ALS 2JQ, UK; 2Unite de Biochimie Physiologique, Universite Catholique de Louvain, Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium

Background and objectives
The plant plasma membrane H+-ATPase (PMA) catalyses the active transport of H+ out of the cell and is responsible for the generation of a pH gradient and a membrane potential across the plasma membrane. These chemical and electrical gradients energize secondary transporters (ion channels or H+-linked carriers) that are important in nutrient uptake, cell elongation, phloem loading and ion homoeostasis. Isoforms of PMA are encoded by a multigene family with members showing tissue- and cell-specific expression and distinct biochemical properties [1]. Sedentary plant-parasitic nematodes such as the cyst-forming (CFN) and root-knot (RKN) nematodes induce the formation of permanent feeding sites within the vascular tissue of infected roots which will supply the developing and adult nematodes with nutrients. The mature feeding sites of both groups are multinucleate large transfer cells, a syncytium in case of the CFN (e.g. Globodera tabacum) and giant cells in the case of RKN (e.g. Meloidogyne incognita) [2]. Very little is known of how these feeding sites are induced and formed. Nutrients and assimilates may be delivered to the feeding sites from the phloem, and the driving forces for the transport processes involved in the uptake to the feeding sites are probably provided by a member of the PMA gene family. In this study the spatial and temporal expression of eight PMA genes of tobacco (Nicotiana plumbaginifolia) after infection with nematodes has been investigated.

Materials and methods
Eight transgenic tobacco (N. tabacum) lines were produced. Each was transformed with a construct containing one of eight available pma transcription promoters of N. plumbaginifolia linked to the reporter gene gusA. The screening for PMA induction in nematode-infected tobacco was done in soil and in vitro. The soil test, performed twice, was done with 9-cm-long plastic tubes, each filled with sandy loam. One seedling was planted in each tube, with four replicates for each pma transformant. The 3-week-old seedlings were infected either with 100 second-stage juveniles (J2) of M. incognita or with three cysts of G. tabacum. GUS expression was tested after 1 month by a histochemical assay. The in vitro test was done in monoxenic cultures on Knop medium (1.5% sucrose; 0.8% agar) at 18 h light/day. 2-week-old seedlings (two seedlings per plate) were inoculated with about 30 surface-sterilized J2 of M. incognita or G. tabacum. GUS expression was tested as above, every other day for M. incognita for a week and after 1 month for both nematode species.

Results and conclusions
The giant cells of M. incognita feeding sites were always strongly GUS stained in the pma4 transformant, even soon after their induction. Weaker GUS staining was also seen with pma1, 2 and 3, but none was found with pma5, 6, 8 and 9. Pma2 transformants demonstrated high GUS activity in root tips soon after invasion by M. incognita but this was not linked to feeding site induction. In contrast, GUS activity in the syncytium formed by G. tabacum was found only with pma4 transformants. Our results indicate that only certain PMA isoforms are involved in feeding site formation (pma4 for both nematode groups; pma 1, 2 and 3 to a lesser extent and for RKN only).

References
1. Michelet B, Boutry M, 1995. Plant Physiology 108, 1-6.
2. Jones MGK, Northcote DH, 1972. Protoplasma 75, 381-395.