A Single-Nucleotide Deletion in the Transcription Factor Gene bcsmr1 Causes Sclerotial-Melanogenesis Deficiency in Botrytis cinerea

Abstract

Botrytis cinerea is an important plant pathogenic fungus with a wide range of host. It usually produces black-colored sclerotia (BS) due to deposition of 1,8-dihydroxynaphthalene melanin in sclerotial melanogenesis. Our previous study (Zhou et al., 2018) reported six B. cinerea isolates producing orange-colored sclerotia (OS) with deficiency in sclerotial melanogenesis. Comparison of ecological fitness (conidia, mycelia, sclerotia), natural distribution, and melanogenesis of selected BS and OS isolates suggests that sclerotia play an important role in the disease cycle caused by B. cinerea. However, the molecular mechanism for formation of the OS B. cinerea remains unknown. This study was done to unravel the molecular mechanism for the sclerotial melanogenesis deficiency in the OS isolates. We found that all the five sclerotial melanogenesis genes (bcpks12, bcygh1, bcbrn1/2, bcscd1) were down-regulated in OS isolates, compared to the genes in the BS isolates. However, the sclerotial melanogenesis-regulatory gene bcsmr1 had similar expression in both types of sclerotia, suggesting the sclerotial melanogenesis deficiency is due to loss-of-function of bcsmr1, rather than lack of expression of bcsmr1. Therefore, we cloned bcsmr1 from OS (bcsmr1^OS ) and BS (bcsmr1^BS ) isolates, and found a single-nucleotide deletion in bcsmr1^OS . The single-nucleotide deletion caused formation of a premature stop codon in the open reading frame of bcsmr1^OS , resulting in production of a 465-aa truncated protein. The transcription activity of the truncated protein was greatly reduced, compared to that of the 935-aa full-length protein encoded by bcsmr1^BS in the BS isolates. The function of bcsmr1^OS was partially complemented by bcsmr1^BS . This study not only elucidated the molecular mechanism for formation of orange-colored sclerotia by the spontaneous mutant XN-1 of B. cinerea, but also confirmed the regulatory function of bcsmr1 in sclerotial melanogenesis of B. cinerea.

Keywords: Botrytis cinerea; bcsmr1; orange-colored sclerotia; sclerotial melanogenesis; single-nucleotide deletion.

Figure 1

Relative expression values of six melanogenic enzyme-coding genes (bcpks12/13, bcygh1, bcbrn1/2, and bcscd1) in the sclerotia of B. cinerea isolates. The sclerotial primordia, immature sclerotia, and mature sclerotia of each isolate were collected from 6-, 8-, and 15-day-old PDA cultures (20°C), respectively. The results are the representative of three independent tests. Bars indicate standard deviations of the means. In each graph, “*” at P < 0.05 and “**” at P < 0.01 between the OS and BS isolates (as two groups) according to Student's t-test.

Figure 2

Comparison of the nucleotide sequences of the transcription factor gene bcsmr1 in Botrytis cinerea and the amino acids sequences encoded by bcsmr1. (A) A schematic diagram showing the structure of bcsmr1 and alignment of the partial DNA sequences of bcsmr1 in different isolates. Nucleotide positions for the exons and introns are indicated. OS, orange-colored sclerotia, BS, black-colored sclerotia. The symbols “∇” and “*” indicate the single-nucleotide deletion and the in-frame stop codon, respectively; (B) Alignment of the deduced amino acid sequences of BcSMR1 in different isolates. The symbol “–” in each column indicates the identical amino acids. Note difference in length of BcSMR1 in the OS and BS isolates, 465 aa long for BcSMR1 in the OS isolates, whereas 935 aa long for BcSMR1 in the BS isolates. NLS, Nuclear Localization Signaling region.

Figure 3

Western blotting detection of the E. coli proteins encoded by bcsmr1^BS and bcsmr1^OS using the anti-His tag antibody. The treatments “bcsmr1-BS”and “bcsmr1-OS” represent the proteins encoded by bcsmr1^BS, and bcsmr1^OS, respectively. The treatment “pET28a” represents the protein encoded by the blank plasmid pET28a alone without bcsmr1^BS or bcsmr1^OS. M = PageRuler^TM prestained protein ladder.

Figure 4

Relative expression values of the transcription factor gene bcsmr1 in the sclerotia of B. cinerea. The sclerotial primordia, immature sclerotia, and mature sclerotia of each isolate were collected from 6-, 8-, and 15-day-old PDA cultures (20°C), respectively. The results are the representative of the three independent tests. Bars indicate standard deviations of the means. “*” at P < 0.05 and “**” at P < 0.01 between the OS and BS isolates (as two groups) according to Student's t-test.

Figure 5

Transcription activation of bcsmr1 in Saccharomyces cerevisiae AH109. (Top) Three schematic diagrams depicting the experimental principle. AD_GAL4 and BD_GAL4 stand for the GAL4 activation domain and the GAL4 DNA-binding domain, respectively. BcSMR1^BS and BcSMR1^OS stand for the proteins encoded by bcsmr1^BS from the BS isolate B05.10 and bcsmr1^OS from the OS isolate XN-1, respectively. BcACTA stands for the protein encoded by bcactA from B05.10. BcSMR1^BS, BcSMR1^OS, and AD_GAL4 can initiate transcription of the reporter genes HIS3, ADE2, and MEL1; (Bottom) Difference between bcsmr1^BS and bcsmr1^OS in transcription activity. Compared to AD_GAL4, BcSMR1^BS initiated strong gene transcription, whereas BcSMR1^OS initiated weak gene transcription. Growth of the yeast mutants on SD/-Trp (left panel, 30°C, 1 day) indicates complementation of the auxotrophic mutant AH109. Growth of the yeast mutants on SD/-Trp-His (right panel, 30°C, 1 day) and production of blue color on the yeast colonies in the presence of X-α-Gal indicate the positive activity of gene transcription.

Figure 6

Colony morphology of Botrytis cinerea isolates B05.10 (BS), XN-1 (OS) and two complementation mutants of XN-1 (XN-BS11, XN-BS14) on potato dextrose agar after incubation at 20°C for 35 days. Note color of the sclerotia formed by different isolates. The arrow mark indicates sporogenic germination on the sclerotia.

Figure 7

Relative expression values of the six melanogenic enzyme-coding genes (bcpks12/13, bcygh1, bcbrn1/2, bcscd1) in the sclerotia of isolate XN-1 of Botrytis cinerea, two complemented mutants of XN-1 (XN-BS11, XN-BS14) and B05.10. The sclerotial primordia, immature sclerotia, and mature sclerotia of each isolate were collected from 6-, 8-, and 15-day-old PDA cultures (20°C), respectively. The results are the representative of three independent tests. Bars indicate standard deviations of the means. ^*P < 0.05 and ^**P < 0.01 (Student's t-test) between each mutant and XN-1; P < 0.05 and P < 0.01 (Student's t-test) between each mutant and B05.10.