Suppressive subtractive hybridization reveals different gene expression between high and low virulence strains of Cladosporium cladosporioides
Introduction
Cladosporium cladosporioides (C. cladosporioides) is a ubiquitous and dematiaceous fungus present in nature, commonly found on dead organic substances and in the air. It causes infections in humans, animals, and plants under certain conditions [1].
The first case of a pulmonary fungus ball produced by C. cladosporioides was reported in 1975 [2]. Since then, more cases of infection have been reported, including subcutaneous mycosis [3] and keratomycosis [4] in humans, mycotic encephalitis and nephritis in a canine [5], and a skin infection in the giant panda [6]. C. cladosporioides also causes various diseases in plants, such as citrus fruit rot, citrus sooty moulds [7], mango sooty blotch disease [8], leaf mold in the Hong Kong orchid tree (Bauhinia blakeana) [9], and leaf mildew in the flamingo lily (Anthurium andraeanum) [10].
C. cladosporioides infection has been steadily increasing, and the elucidation of the pathogenic and molecular mechanisms involved is critical in order to better treat the infections and infection-associated diseases. In our previous work, a mutant strain named Zt was obtained from the wild-type C. cladosporioides strain Z20 [11]. The Zt mutant produces less spores and the color of its colony is lighter than the wild type. More importantly, it is less virulent to both citrus plants and mice.
Suppression subtractive hybridization (SSH) is a technique that allows for PCR amplification of only the cDNA fragments that differ between the control (driver) and the experimental (tester) groups [12]. This method has been employed previously to identify strain-specific DNA sequences in other fungal species, for example, genes associated with morphology in Aspergillus niger [13], and genes associated with phase switch (from mycelial phase to yeast phase) in Penicillium marneffei [14]. SSH has also been employed to identify differences in gene expression between high and low virulent strains of Flavobacterium columnare [15].
In order to better understand the molecular mechanism of C. cladosporioides infection, SSH was used in this study to compare the differences in gene expression between the highly virulent Z20 strain and the lowly virulent Zt mutant. Our study shed new light on the mechanism of C. cladosporioides infection and may help prevent and treat its associated diseases.
Section snippets
Fungal strains and culture conditions
The C. cladosporioides Z20 strain (Genebank accession: JQ727688.1) used in this study is a highly virulent strain[6]. The C. cladosporioides mutant strain Zt (Genebank accession: KR084328.1) was derived via mutagenesis with microwave and N-Methyl-N’-nitro-N-nitrosoguanidine (NTG) treatment [11]. The Zt strain is less conidial, sporulates slower and ages earlier. Its colony color is much lighter than Z20 and its infection rate to citrus and mice is significantly lower [11]. Both strains were
Identification of subtractive library
PCR analysis showed that the cDNA inserts from the forward and reverse libraries ranged from 100 to 500 bp in size, and insert efficiency was greater than 95% (Fig. 1). This indicates that the SSH libraries were constructed successfully.
Sequence analysis of expressed sequence tags (ESTs)
Sequencing analysis showed that 204 and 206 ESTs were obtained from the forward and reverse SSH libraries, respectively. After eliminating ESTs with a length of less than 100 bp, vectors and primers, 119 ESTs were obtained and 61 unigenes were assembled from the
Discussion
In this study, SSH was used to compare the gene expression between a high virulence strain (Z20) and low virulence strain (Zt) of C. cladosporioides. A total of 61 unigenes from the forward library and 42 from the reverse library were obtained.
In the forward library, many pathogen and growth-associated genes were identified. The MIP1 gene (Table 2, F117) was up-regulated more than 300 times in Z20 strain than in Zt strain. It encodes a target of rapamycin complex 1 (TORC1) subunit, which
Conclusions
In this study, we compared the differences of gene expression between two C. cladosporioides strains: the highly virulent Z20 and less virulent Zt strain. A total of 103 differentially-expressed unigenes were identified, including 61 in Z20 strain and 42 in Zt strain. Functional analysis revealed that the 103 unigenes were mainly associated with metabolic processes, enzyme activity, response to stimulus/signaling, establishment of localization and transporters. The qRT-PCR analysis of 12
Acknowledgments
This study was supported by grants from the National Key Technology R&D Program (2012BAC01B06), the Applied Basic Research Project in SiChuan Province (2013JY0175), and the international cooperation funds of giant panda (AD1415). All authors have agreed to submit this manuscript to the “Microbial Pathogenesis”.
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Identification and characterization of sex related genes in Actinidia arguta by suppression subtractive hybridization
2018, Scientia HorticulturaeCitation Excerpt :A total of 678 up-regulated unigenes, 309 in female and 369 in male respectively, were identified by SSH method, indicating a large number of genes may involve in sex differentiation of Actinidia arguta. These unigenes were categorized into many groups according to GO classification, among which the dominant GO terms (cell and cell part in cellular component category; catalytic activity and binding in molecular function category; metabolic process and cellular process in biological process category) were identical with some other SSH libraries (Miao et al., 2015; Yang et al., 2015; Gu et al., 2016), although these libraries aimed at different biological researches. As the genes belong to these GO terms are essential for vital activities of plants, variant transcription of these genes will lead to different phenotypes and physiological stages as well as response to biotic and abiotic stress.