MRP3 is a sex determining gene in the diatom Pseudo-nitzschia multistriata

Abstract

A broad diversity of sex-determining systems has evolved in eukaryotes. However, information on the mechanisms of sex determination for unicellular microalgae is limited, including for diatoms, key-players of ocean food webs. Here we report the identification of a mating type (MT) determining gene for the diatom Pseudo-nitzschia multistriata. By comparing the expression profile of the two MTs, we find five MT-biased genes, of which one, MRP3, is expressed exclusively in MT+ strains in a monoallelic manner. A short tandem repeat of specific length in the region upstream of MRP3 is consistently present in MT+ and absent in MT- strains. MRP3 overexpression in an MT- strain induces sex reversal: the transgenic MT- can mate with another MT- strain and displays altered regulation of the other MT-biased genes, indicating that they lie downstream. Our data show that a relatively simple genetic program is involved in defining the MT in P. multistriata.

Fig. 1

P. multistriata MT-biased genes conservation. a Conservation in diatom genomes. The diatom species with a sequenced genome are shown in a simplified diatom phylogenetic tree based on 18S (courtesy of W.H.C.F. Kooistra, modified from ref. ), color in the pie next to each species indicates the presence of a homolog, white indicates that no homology had been detected. Shades on the tree demarcate diatom lineages indicated on the left. The red rectangle indicates the area that is magnified in b. b Conservation in the transcriptomes of Pseudo-nitzschia and Fragilariopsis species

Fig. 2

MRP3 alleles. a Schematic representation of the genomic region between 91,500 and 94,000 bp on scaffold 147. The intronless gene MRP3 is represented as a thick arrow with the CDS in yellow and the UTR regions in orange, the transcription start site (TSS), the start codon (ATG) and the stop codon (TAG) are indicated. Primers are represented as black arrows. The purple box represents CTA repeats and the black box a stretch of Ns. b Electrophoretic gel with amplicons obtained from five MT+ and three MT− strains. The high band (A, orange arrow) is present in MT+ only, m stands for marker. c Electrophoretic gel with amplicons obtained using primers F147p3-R147p2 from four MT+ strains which, with the PCR shown in b, had only the A band (LV89 and LV130), or the A and the M band (LV121 and B856). d Electrophoretic gel with amplicons obtained from the same four MT+ strains as in c, using a different reverse primer. e Schematic representation of the A, M, B, and N alleles. The purple, brown and violet boxes represent CTA repeats, GTA repeats and GGA repeats respectively, the number of repetitions is indicated for each triplet. The pink box represents a repetitive sequence, the blue box remnants of a transposase. The sequence in the gray box could not be amplified

Fig. 3

Monoallelic expression of MRP3 is linked to the A allele. a IGV (Integrative Genomics Viewer) visualization of the MRP3 gene on scaffold 147. Colored vertical lines at the bottom represent the DNA nucleotides in the genome, with SNPs indicated below. The gray peaks above correspond to the RNA-seq reads from strain B856 (MT+, A/M genotype). Colored bars are in correspondence of nucleotides that differ between the reference DNA sequence and the transcript sequence. b The top electropherogram shows the sequence of an MRP3 fragment amplified from the gDNA of strain LV149 (MT+, A/M genotype), the bottom track is from the cDNA of the same strain. A black arrow indicates the double peak in the gDNA. c Schematic representation of the alignment of Sanger sequences from PCR fragments obtained amplifying either gDNA or cDNA, or from different TOPO vectors in which alleles were cloned. The strain name, its genotype and the type of sequence are shown on the left. The consensus sequences of the A, M, and B alleles were included in the alignment for reference. A schematic representation of the genomic region under study is shown above the alignment (see Fig. 2a), with the primers used for sequencing indicated. Polymorphic nucleotides or indels are indicated at their approximate position. Note that the MRP3 transcript shows a C, which is on the A allele, while T, the alternative nucleotide in that position, is on the M allele

Fig. 4

Sex reversal in the MT- LV136 transformed strain overexpressing MRP3. a On the top, amplification of a fragment of the nourseothricin resistance gene (primers NATfor/NATrev, left) and a fragment of the transgene from the H4 promoter to the MRP3 CDS (primers H4up1-0020770+R, right) from the genomic DNA of strain LV136T3 is shown; on the bottom, amplification of a fragment of the MRP3 transcript (primers 0020770+F/0020770+R for MRP3) from the cDNA of strain LV136T3 is shown. m indicates the 100 bp DNA ladder. b Images of crosses of wild-type LV136 (MT−) and transformant LV136T3 with LV168 (MT+) and MC1217-17 (MT−). Wild-type cells mated with LV168 producing initial cells (the longest cells indicated by arrows), while the transformant cells did not. Opposite results were observed in crosses with MC1217-17. Scale bar = 20 μm. c PCR analysis for the variable region of the MRP3 alleles on gDNA of F1 siblings obtained from the cross between MC1217-17 (MT−) and LV136T3. 1075-25 is a control MT+ strain. d, e The LV136T3 strain displays amplification of MT+ specific genes (top two gels) and less intense signals for MT− specific genes with respect to the nontransformed LV136 strain (bottom gel and qPCR graphs). Amplification of an intron-containing CDK-A fragment is shown to demonstrate that the cDNAs are free from genomic contamination. In the qPCR graphs, expression levels of LV136T3, of the MT+ control strain 41.6 and of LV136 are relative to expression levels of an MT− control (strain 41.4) set as 0 (see Methods). Error bars represent the standard deviation of three biological replicates