Kim Hyojeong, Metzenberg Robert L, Nelson Mary Anne
Department of Biology, The University of New Mexico, Albuquerque, New Mexico 87131, USA.
Eukaryot Cell. 2002 Dec;1(6):987-99. doi: 10.1128/EC.1.6.987-999.2002.
A putative pheromone precursor gene of Neurospora crassa, mfa-1 (which encodes mating factor a-1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high mfa-1 expression in all mating type a tissues and suggested low expression levels in mat A tissues. The mfa-1 gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3' untranslated region (3' UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of Sordaria macrospora and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were mfa-1 mutants with intact ORFs but multiple mutations in the 3' noncoding region (CAAX mutants). The 3' UTR is required for the full range of mfa-1 gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 micro M] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous mfa-1 crosses. As males, YAAX mat a mutants were unable to attract wild-type mat A trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX mat a mutants were able to mate and produce sexual progeny despite their inability to attract mat A trichogynes. In the mat A background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the mfa-1 gene appears to have multiple roles in N. crassa development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by mat a to attract trichogynes of mat A; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.
粗糙脉孢菌的一个假定信息素前体基因mfa-1（编码交配因子a-1），被鉴定为饥饿菌丝体和子囊壳cDNA文库中丰度最高的克隆。Northern分析表明，mfa-1在所有a交配型组织中高表达，而在A交配型组织中表达水平较低。mfa-1基因表达为一个约1.2kb的转录本，预计编码一个24个残基的肽段，后面跟着一个长的3'非翻译区（3'UTR）。预测的MFA1序列与大孢粪壳菌的PPG2显示100%的序列同一性，并且与许多疏水性真菌信息素前体在结构上相似（羧基末端CAAX基序）。分离出了开放阅读框（ORF）被破坏的突变体，其中关键的半胱氨酸残基已变为不可异戊二烯化的残基酪氨酸（YAAX突变体），以及ORF完整但3'非编码区有多个突变的mfa-1突变体（CAAX突变体）。3'UTR是mfa-1基因全部活性所必需的。这两类突变体都表现出延迟和减缓的营养生长（补充微量[30μM]的鸟氨酸、瓜氨酸或精氨酸可抑制这种生长），以及异常的有性发育。当作为母本与野生型父本杂交时，CAAX和YAAX突变体的子囊孢子产量大幅降低。纯合mfa-1杂交中不产生子囊孢子。作为父本，YAAX a交配型突变体无法吸引野生型A交配型的受精丝（雌性特异性菌丝）或启动有性发育，而CAAX a交配型突变体尽管无法吸引A交配型的受精丝，但仍能够交配并产生有性后代。在A交配型背景下，CAAX和YAAX突变体均表现出正常的雄性育性，但营养生长有缺陷且雌性有性发育异常。因此，mfa-1基因似乎在粗糙脉孢菌的发育中具有多种作用：（i）它编码一种疏水性信息素，其羧基末端半胱氨酸残基可能被法尼基化和羧甲基化，a交配型需要它来吸引A交配型的受精丝；（ii）它参与两种交配型的雌性有性发育和子囊孢子产生；（iii）它在两种交配型的营养生长中发挥作用。
