Chang Perng-Kuang
Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Allen Toussaint Boulevard, New Orleans, LA 70124, USA.
J Fungi (Basel). 2024 Oct 16;10(10):719. doi: 10.3390/jof10100719.
Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the gene at the DNA and transcript levels affected the survival, growth, and development of . Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops.
真菌HacA/Hac1转录因子在调节未折叠蛋白反应（UPR）中起关键作用。UPR有助于细胞维持内质网（ER）蛋白稳态，这对生长、发育和毒力至关重要。该基因编码一个富含碱性和酸性氨基酸的结构域（Bsc）和一个碱性亮氨酸拉链（bZip）结构域，并具有一个非常规内含子（Nt20）。在本研究中，利用CRISPR/Cas9剖析Bsc编码序列、bZip编码序列和Nt20序列，以阐明基因型与表型之间的关系。在Bsc和bZip实验组中，两个编码序列中观察到的所有突变都是框内的，这表明框外突变是致死的。Nt20实验组中转化体的存活率很低，约为7%。内含子中的突变主要由框外插入和缺失组成。除了类似野生型的分生孢子形态外，突变体还表现出多种菌落形态，包括菌核形态、混合形态（分生孢子和菌核）和菌丝形态。使用二硫苏糖醇进行的内质网应激试验表明，菌核和菌丝突变体比分生孢子突变体敏感得多。此外，菌丝突变体不能产生黄曲霉毒素，但仍能产生曲酸和 kojic 酸。针对包含Bsc和bZip的区域进行的RNAi实验表明，转化体存活率普遍下降，少数转化体表现出表型变化。该基因在DNA和转录水平上的缺陷影响了其存活、生长和发育。因此，该基因可能是未来宿主诱导基因沉默策略的一个有前景的靶点，旨在控制感染并减少作物中的黄曲霉毒素污染。
