Institut Pasteur, INRA, Unité Biologie et Pathogénicité Fongiques, Paris, France.
Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.
mSphere. 2019 Feb 13;4(1):e00709-18. doi: 10.1128/mSphere.00709-18.
The heterozygous diploid genome of is highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-I endonuclease and (ii) detection of the resulting long-range homozygosis. I-I successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whose ortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy of rescued cells homozygous for the right arm of Chr7B, demonstrating that the RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provide with increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species. is a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such as By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 isolates, we were able to ascribe the chromosome 7 homozygosis bias of the laboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in the gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS in could favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.
是一种重要的真菌病原体，其繁殖方式主要为无性繁殖。它的基因组对重排具有高度的耐受性，特别是频繁发生的杂合子二倍体的杂合性丢失（loss of heterozygosity，LOH）事件。在实验室菌株 SC5314 中，虽然 LOH 事件普遍存在，但某些染色体存在着染色体纯合性偏向，即只有两个同源物中的一个能够处于纯合状态。这表明存在隐性致死等位基因（recessive lethal allele，RLA），阻止了这些染色体上大规模 LOH 事件的稳定维持。为了验证第 7 号染色体（Chr7）上存在 RLA，我们利用了一种系统，该系统允许（i）I-I 内切酶在 Chr7 上诱导 DNA 双链断裂（double-strand break，DSB），（ii）检测由此产生的长距离纯合性。I-I 成功地在 Chr7 的两个同源物上诱导了 DSB，通常通过基因转换进行修复。值得注意的是，没有回收到 Chr7B 右臂纯合的细胞，这证实了该区域存在 RLA(s)。对 RLA 候选基因进行基因组数据挖掘，在 C7_03400c 的 HapB 等位基因内发现了一个提前产生无义的单核苷酸多态性（single nucleotide polymorphism，SNP），其直系同源物编码必需的 Mtr4 RNA 解旋酶。用野生型的 基因进行互补，拯救了 Chr7B 右臂纯合的细胞，证明了 基因的 RLA 是导致 SC5314 菌株 Chr7 纯合性偏向的原因。此外，我们观察到 Chr7 上的主要重复序列（major repeat sequences，MRS）作为同源重组热点。这种重组事件为 提供了更多的机会来存活 DNA DSB，其修复可能导致隐性致死或有害等位基因的纯合性。这可能解释了 MRS 在该物种中的维持。
