ANALYSIS OF PECTINOLYTIC ENZYMES AND RELATED GENES IN PYRENOPHORA SPP.

Pyrenophora graminea Ito e Kuribayshi and P.teres (Died.) Drechsl. respectively cause leaf stripe and net blotch on barley leaves. Both pathogens are seed-borne and can systemically colonize their host. In contrast to P.teres, P. graminea cannot successfully cause disease by direct leaf infection. Since in the most part of their life cycle both pathogens show an intercellular mode of growth in host tissue, it was assumed that enzymes degrading pectic components of plant cell walls are involved in colonization. Previous results showed in Pyrenophora graminea cultured in several media containing pectin high a pectinolytic activity revealed by enzymatic assay while a faint expression of endopolygalacturonase (endo-PG) in Northern blot when RNAs were probed to a heterologous gene (clone pCC2 derived from Fusarium moniliforme). Further by PCR experiments in Pyrenophora graminea (strain I2-88) and Pyrenophora teres (I4-88) a genomic region between specific primers (inferred by the Fusarium moniliforme endo-PG gene sequence) has been amplified in both species: Pg2-PCR and Pt4-PCR respectively. Both PCR product were 1.5Kbp. Pg2-PCR has been used as a homologous probe in Northern blot experiments on P.graminea and P.teres RNAs in vitro induced by pectin or barley cell walls, compared to control RNAs. A strong expression has been detected in both fungi although it was constitutive and the transcript was at a lower mw (about 1Kbp) compared to the previous one recognized by pCC2 in P.graminea and to the one identified in F.moniliforme. Furthermore the probe Pg2-PCR apparently belongs only to Pyrenophora spp. genome since it does not recognize Fusarium moniliforme sequences neither in Northern blot nor in Southern blot experiments. Further analysis will focus on Pg2-PCR cloning and sequencing to identify both structure and function of the relative gene. A concomitant study about P.graminea secreted polygalacturonase activity showed a very high endo-PG activity in cultures containing pectin, an about ten-fold lower activity in cultures containing cell walls and only barely detectable activity in control cultures. Isoelectrofocusing patterns were equivalent in both inductive systems. The two main bands (42 kD and 40 kD, Ip about 8.5 and 7.5 respectively) were recognized in Western blot by a Fusarium moniliforme anti-PG polyclonal antibody. What is the significance of different results obtained by those two approaches is still an open question: on one side a constitutive gene function identified only by the homologous probe Pg2-PCR, on the other side an inducible activity identified by the heterologous antibody. Multiple forms of PG may exist in Pyrenophora spp. with different expression modes. It is noteworthy that both approaches produce equivalent results with two inductive systems (pectin and cell walls) suggesting a role of the same PGs in host-pathogen interaction. Pyrenophora graminea Ito e Kuribayshi and P.teres (Died.) Drechsl. respectively cause leaf stripe and net blotch on barley leaves. Since both pathogens show an intercellular mode of growth in part of their life cycle, pectinolitic enzymes could be important for host colonization. A 1.5 Kbp PCR product (Pg2-PCR), previously obtained from P.graminea with specific primers from Fusarium moniliforme endo-PG gene sequence, has been used as a homologous probe in Northern blot experiments.