Factors that control antifungal activity of tobacco PR -5 protein, osmotin
Abstract
Osmotin is a pathogenesis-related (PR) protein, family 5, that accumulates in tobacco cells in response to pathogen attack and osmotic stress. The accumulation of osmotin and other PR-proteins is believed to play a role in the plant disease defense response by limiting pathogen infection. To understand how osmotin functions we, (1) constructed chimeric proteins of osmotin to determine which domains confer antifungal activity, (2) used transpon-mutagenesis of an osmotin sensitive yeast to determine components responsible for osmotin cytotoxicity, and (3) demonstrated functional conservation of cell wall proteins as determinants for osmotin cytotoxicity in yeast and Fusarium oxysporcan. Five domain-swapped osmotin proteins were constructed, each of which contained a homologous domain of the less active, PR-5 antifungal protein, A9. Recombinant proteins, native osmotin and A9 were expressed in Nicotiana benthamiana using a tobacco mosaic virus (TMV) vector-system, purified from extracellular fluid extracts, and assayed for their toxicity to the yeast S. cerevisiae. We cautiously propose that the fourth domain (the second loop) may play an important role in interacting with yeast cell wall for the antifungal activity. ore16/ore19 (osmotin resistant mutant) was isolated from screening of a transposon-mutagenized osmotin sensitive yeast BWG7a library. ore16/ore19 is a mutation in TTP1/MNN2, a α-1,2-mannosyltransferase, which catalyzes the addition of the first mannose to the N-linked mannans on the yeast cell wall. Together with studies on MNN4, MNN6, we showed that phosphomannoproteins bind osmotin and act as facilitators of osmotin cytotoxicity. PIR2, a yeast cell wall protein responsible for osmotin resistance, was transformed into F. oxysporum. PIR2 transformants (1) contained PIR2 protein correctly localized on the cell wall, (2) were 2 fold more tolerant to osmotin than wild type or vector transformants and, (3) had increased virulence against 3 week-old tobacco seedlings. Three FOR (Fusarium Osmotin Resistance) genes were isolated from screening of a F. oxysporum cDNA library for genes conferring osmotin resistance to yeast. Sequence analysis indicates these genes encode cell wall proteins or proteins involved in cell wall synthesis/regulation suggesting cell wall components are important factors in osmotin cytotoxicity and are conserved in both filamentous fungus and yeast.
Degree
Ph.D.
Advisors
Bressan, Purdue University.
Subject Area
Molecular biology|Botany
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