Molecular cloning of the barley pathogen induced
gene coding for "Thaumatin like" protein
Giampiero Valè, Emanuela Torrigiani, Hala Toubia-Rahme
and Giovanni Delogu

Istituto sperimentale per la Cerealicoltura Sezione di
Fiorenzuola d'Arda - Via S. Protaso 302 - I-29017 Fiorenzuola
d'Arda (PC), Italy


In Northern and Central Italy the infection of seeds by Pyrenophora graminea leads to a severe yield reduction (Porta-Puglia et al., 1986; Delogu et al., 1989). An evaluation of physiological, biochemical and virulence characteristics of different isolates collected in various Italian barley-growing areas revealed for such characters a broad variability (Gatti et al., 1992).

The inoculation of barley rootlets with Pyrenophora graminea isolates activates a specific set of plant's genes; Northern blot analysis showed accumulation of at least three mRNAs belonging to peroxidase, thaumatin-like and thionin gene families as response to inoculation (Valè et al., 1994). The expression of mRNAs corresponding to thaumatin-like gene was the most aboundant and was detected in all the cultivar/isolate combination tested, even if the level of the corresponding transcript accumulated in the cells was depending from the isolate used for inoculation. To detect the expression of gene encoding for thaumatin-like proteins a cDNA isolated from wheat leaves inoculated with Erisiphe graminis f.sp. hordei (Rebmann et al., 1991), was used as molecular probe. We have therefore decided to clone the corresponding barley cDNA. A cDNA was synthetized from mRNAs isolated from barley rootlets of cv. Thibaut inoculated for 24h with the incompatible isolate I2 of P. graminea using a 16 mer primer (5' TGGACAGAAGGTGATC 3') designed on the basis of the 3' end sequence of the corresponding wheat cDNA clone (Rebmann et al., 1991). The cDNA obtained was amplified with the "polymerase chain reaction" using a degenerated primer (5' ATGGCC(TACG)AC(TACG)TT(TC)AA(TC)AT(TCA) 3') designed on the basis of the N-terminal aminoacid sequence of the protein "Thaumatin-like" Hv1 purified from barley leaf inoculated with E. graminis f. sp. hordei (Bryngelsson and Gréen, 1989). The sequence analyses of the btl12 (barley thaumatin like) cDNA clone showed a high degree of homology at nucleotide and amino acid level with the sequence of the wheat thaumatin-like protein (pWIR232) (Rebmann et al., 1991). In figure 1 the best alignment of the barley and wheat thaumatin-like proteins is shown.

Figure 1. The best alignment of barley and wheat thaumatin-like proteins is shown.

BTL12- MAT-------------------FNIKNNCGSTIWPAGIPVGGGFELGSGQ-31
       III                   IIIIIIII IIIIIIIIIIIII IIIII
WIR232-MATSPVLFLLLAVFAAGASAATFNIKNNCGFTIWPAGIPVGGGFALGSGQ-50

BTL12- TSSTNVPAGTQAGRIWARTGCSFNGGTGSCQTGDCGGQLSCSLSGQPPAT-81
       III IIIIIIIIIIIIIIIIIIIIIIxIIIIIIIIIIIIIIIIII IIII
WIR232-TSSINVPAGTQAGRIWARTGCSFNGGSGSCQTGDCGGQLSCSLSGRPPAT-100

BTL12- LAEFTIGGGSTQDFYDISVIDGFNLAMDFSCSTGDALQCRDPSCPPPQAY-131
       IIIxIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
WIR232-LAEYTIGGGSTQDFYDISVIDGFNLAMDFSCSTGDALQCRDPSCPPPQAY-150

BTL12- QHPNDVATHACSGNNNYQITFCP-154
       IIIIIIIIIIIIIIIIIIIIIII
WIR232-QHPNDVATHACSGNNNYQITFCP-173
The character "I" shows that two aligned residues are identical.
The character "x" shows that two aligned residues are similar.


References

Bryngelsson, T., and B. Gréen. 1989. Characterization of a pathogenesis-related, thaumatin-like protein isolated from barley challenged with an incompatible race of mildew. Physiol. Mol. Plant Pathol. 35: 45-52.

Delogu, G., A. Porta-Puglia and G. Vannacci. 1989. Resistance of winter barley varieties subjected to natural inoculum of Pyrenophora graminea. J. Genet. & Breed. 43: 61-66.

Gatti, A., F. Rizza, G. Delogu, V. Terzi, A. Porta-Puglia and G. Vannacci. 1992. Physiological and biochemical variability in a population of Drechslera graminea. J. Genet. & Breed. 46: 179-186.

Porta-Puglia, A., G. Delogu and G. Vannacci. 1986. Pyrenophora graminea on winter barley seed: effect on disease incidence and yield losses. J. Phytopathol. 117: 26-33.

Rebmann, G., F. Mauch and R. Dudler. 1991. Sequence of a wheat cDNA encoding a pathogen-induced thaumatin-like protein. Plant Mol. Biol. 17: 283-285.

Valè, G.P., E. Torrigiani, A. Gatti, G. Delogu, A. Porta-Puglia, G. Vannacci and L. Cattivelli. 1994. Activation of genes in barley roots in response to infection by two Drechslera graminea isolates. Physiol. Mol. Plant Pathol. 44: 207-215.