Inhibition of Neurospora crassa growth by a glucan synthase-1 antisense construct.
We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)beta-glucan synthase [E.C. 2.4.1.34; UDPglucose: 1,3-beta-D-glucan 3-beta-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)beta-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2[qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction ingrowth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)beta-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)beta-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents.
Tentler
S
S
MycoTox, Incorporated, 4200 East Ninth Ave, Box B-111, Denver, CO 80262, USA.
Palas
J
J
Enderlin
C
C
Campbell
J
J
Taft
C
C
Miller
T K
TK
Wood
R L
RL
Selitrennikoff
C P
CP
D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018390:Gene Targeting; D005800:Genes, Fungal; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008565:Membrane Proteins; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D012331:RNA, Fungal / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D014170:Transformation, Genetic
D000935:Antifungal Agents; D017931:DNA Primers; D008565:Membrane Proteins; D016372:RNA, Antisense; D012331:RNA, Fungal; D029702:Schizosaccharomyces pombe Proteins; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase