1. Introduction
China is the largest producer and consumer of fresh grapes in the world. There are reports indicating that after conducting field investigations in the main grape-producing areas of Xinjiang and Hebei, it was found that the grape-planting area of China has remained stable at over 10 million mu over the past five years. The production of grapes in China continued to increase, reaching 13.667 million tons in 2018 and becoming the world’s largest producer. The distribution of grape-planting areas in China is concentrated, with the main production areas located in historically advantageous planting areas, such as Xinjiang, Shanxi, Hebei, and Shandong. The planting areas of five provinces have reached 4.629 million mu, accounting for 42.6% of the total area.
Grape anthracnose, also known as late rot, is an important disease impacting grapes that is mainly caused by the fungus
Colletotrichum species, such as
Colletotrichum gloeosporioides and
Colletotrichum cuspidosporium [
1]. The disease often occurs at the end of grape growing and fruit ripening, which mainly harms fruits and ear rachis of grapes. In addition, it can also damage leaves, new shoots, tendrils, fruit stalks, and other parts. The diseased fruits and leaves generate black-brown anthracnose spots, and the fruit surface is often festered with extravasated juice, which seriously reduces the yield and quality of the grapes. At present, the disease occurs in most grape-producing areas in China, and in recent years,
Colletotrichum aenigma was also reported as a causal agent of Grape anthracnose in China and South Korea [
2,
3,
4,
5].
Peroxisome is a kind of monolayer-coated organelle with active metabolisms and highly dynamic changes in eukaryotes, which is mainly involved in the metabolism of fat, the production and scavenging of reactive oxygen species, etc. Additionally, it has been reported that peroxisome is essential for the pathogenicity of many phytopathogenic fungi, including
Magnaporthe oryzae,
Botrytis cinerea,
Fusarium graminearum, and so on [
6]. However, it is still undocumented whether the peroxisomes function in the infection cycles of
C. aenigma. The peroxisomes were found to be induced and greatly proliferated at the early stages of the conidial germination and infection of
M. orzyae [
7]. Therefore, the study of peroxisome distribution and dynamics in the process of growth, development, and pathogenesis is of great significance in revealing the involvement of peroxisomes in the pathogenicity of phytopathogenic fungi, including
C. aenigma.
Fluorescent protein labeling is an effective method of studying promoter activity, gene expression dynamics, cellular and subcellular localization of proteins, and the growth and development of organisms [
8]. Green fluorescent protein (GFP), first found in
Aequorea victoria in 1962, has stable, intuitive, and convenient fluorescence properties and is the most widely used fluorescent protein at present. Red fluorescent protein (RFP) was isolated from
Discosoma sp. by MATZ et al. in 1999. The different variants of red fluorescent protein show different spectral and physicochemical properties, emitting various colors of fluorescence. For example, mCherry emits 610 nm fluorescence under 587 nm excitation light and cherry-red under a fluorescence microscope. It has the advantages of rapid ripening and good monomer properties and has a wide range of applications. Calcofluor white (CFW) and DAPI are two kinds of dyes that display blue fluorescence under a fluorescent microscope [
9]. CFW can bind cellulose and chitin in the fungal cell wall and is thus widely used for labeling cell walls in fungi. DAPI is a nuclei dye that penetrates the cell membrane and binds specifically to DNA in a non-embedded way [
10]. Histone is a basic DNA-binding protein, which is mainly located in the nucleus. The fusion expression of histone and fluorescent protein can realize the fluorescent labeling of the nucleus and assist in the dynamic observation of the nucleus [
11]. Wang J Y et al. fused the peroxisome localization signal PTS1/PTS2 into GFP, respectively. They then successfully located the peroxisome of
Magnaporthe oryzae [
12]. Fluorescent protein localization was also utilized in
Fusarium oxysporum f. sp. niveum [
13] and
Botrytis cinerea [
6]. We can also study the function of genes and the dynamic changes of nuclei in the process of infection through the location of the peroxisome and the nuclei. However, there is no study on the fluorescence localization of peroxisome in
C. aenigma.In this present work, GFP-PTS1 [
13], DsRED-PTS1 [
13], mCherry-H2B [
14], and PNMCherryA, under different promoters, were used as fluorescent markers to label the peroxisomes and nuclei in
C. aenigma via
Agrobacterium tumefaciens-mediated fungal transformation (
AtMT). Using the fluorescently labeled strains, the peroxisomal dynamics and nuclear distribution in
C. aenigma were detected under a fluorescent microscope. In addition, we obtained a library of T-DNA insertion transformants for mutant selection. Consequently, we provide a tool and basic data for the further study of the organelle biogenesis, fungal growth, development, and pathogenicity of
C. aenigma.
2. Materials and Methods
2.1. Test Strains and Culture Conditions
C. aenigma and its transformants were cultured on the complete medium (CM) [
15] in 28 °C darkness.
Colletotrichum aenigma (Genbank Taxonomy ID: 1215731) strain NH-8 was isolated from a grape vineyard in Ninghai country, Zhejiang province, China, identified by the morphology and the sequences of ITS, GAPDH, and tubulin, and kept in the fungal strain store in Zhejiang Academy of Agricultural Sciences (accession number ZN-05827).
2.2. Plasmids and AtMT
PHMGA, pHMR1 [
16], and pKD9-mCherry-H2B [
17] were introduced into
C. aenigma by AtMT [
18] transformation, and the transformants CA-HMGA, CA-HMR1, and CA-mCherry-H2B were obtained. In the same way, PNMCherryA was introduced into CA-HMGA to obtain a CA-HMGA-PNMCherryA colocalized strain.
A. tumefaciens strains were cultured in a YEB liquid medium containing 50 μg/mL ampicillin, 50 μg/mL rifampicin, and 50 μg/mL kanamycin in 28 °C darkness for 2 days with 180 r/min [
6]. Obtaining
C. aenigma spores which were cultured on CM medium at 28 °C for 5 days, we then prepared the spore suspension into 1 × 10
6/m L. Afterward, the spore liquid and
A. tumefaciens were co-cultured on IM medium [
19] at 23 °C for 2 days. Finally, the transformants were screened on a CM medium plate containing 150 μg/mL hygromycin B.
2.3. Fluorescence Stability and PCR Detection of Transformants
Taking CA-HMR1 transformants as an example to detect genetic stability is shown in
Figure 1. Five CA-HMR1 transformants (T0) with bright fluorescence expression were selected randomly by resistance screening and fluorescence microscopy. Five mycelial blocks were taken from the colonial edges of each of the T0 transformants and subcultured to generate 25 progeny transformants (T1). The fluorescence of each T1 transformant was observed and those with fluorescence were sub-cultured to obtain T2 generation, successively, until the T7 generation. The fluorescence of hyphae and the spores of all the progeny transformants was observed by a fluorescence microscope to analyze the fluorescence stability of the transformants.
The transformants of each generation were detected by PCR to determine whether they contained the hygromycin resistance gene and the corresponding fluorescent protein gene. Genomic DNA was extracted by the CTAB [
20] method. The fragments of hygromycin, GFP, DsRED, mCherry, and G418 genes were amplified by using the primer pairs HPH52/HPH34, GFP1-CHK1/GFP-CHK2, RED1/RED2, RED3/RED4, and NEO1/NEO2, respectively. The genomic DNA of the
C. aenigma wild strain was used as a negative control, and the corresponding plasmid DNA was used as a positive control. The PCR products were detected by electrophoresis with 1.0% agarose gel under 150V for 20 min. The primers used were listed in
Table S1 [
7] and PCR procedures were attached in
Table S2.
2.4. Laser Confocal Observation and Co-Localization with Fluorescent Dyes
The transformants with strong fluorescence expression, clear localization, and stable subculture were selected, and a small number of hyphae and spores were selected to observe and record the fluorescence localization using a laser confocal microscope (ZEISS LSM780).
The red fluorescence was observed at an excitation wavelength of 543 nm and an emission wavelength of 570–630 nm. The green fluorescence was observed at an excitation wavelength of 488 nm and an emission wavelength of 495–550 nm. In addition, the blue fluorescence was observed at an excitation wavelength of 405 nm and an emission wavelength of 410–480 nm.
CFW and DAPI Staining: 10 μL 10 μg/mL CFW staining solution was added to avoid light staining for 5 min, and the blue fluorescence produced by the cell wall was observed [
8]. Using a similar method, 10 μL 50 μg/mL DAPI staining solution was added to observe the blue fluorescence produced by the nucleus [
21].
2.5. Comparison of Phenotype and Spore Production of Transformants
The tested strain was inoculated on the CM medium plate for 3 days, and then the round mycelial blocks with a diameter of 0.5 cm were obtained at the colony edge of the transformant and strain C. aenigma with a hole punch, and finally transferred to the new CM medium plate with 3 repeats for each strain. After 3 days of dark culture at 28 °C, the colony diameter was measured and photographed, and the difference in growth rates was statistically analyzed. The growth rate was calculated with V = (D − d)/T; V is the growth rate (mm/d); D is the average diameter of the colony (mm); d is the diameter of the inoculated plaque (mm); and T is the growth time (d).
To harvest the spores, 4 mL of sterilized distilled water was added to each CM culture plate. Then the colony surface was gently scraped with a disposable coating stick to wash the spores into the water. The spore suspension was filtered with three layers of sterilized lens wipes with 3 repeats for each treatment. The spore concentration was calculated on a Hemocytometer, and the spore generation of each strain was calculated and compared.
2.6. Pathogenicity Test
The pathogenicity was tested by acupuncture inoculation (acupuncture 0.5 cm). The spore suspensions of the transformants and wild strain of C. aenigma were prepared in 106 mL−1. Afterward, 20 fresh grapes and a few leaves were wiped briefly with 75% alcohol, naturally dried, and acupunctured using sterile needles, then they were inoculated by dropping the suspension on the acupunctured sites with pipettes.
The mycelial blocks were also used in inoculation by placing them on the acupuncture site of the leaves. The sterile water was used as the control, and a group of non-invasive inoculation groups was set up. The treatment was repeated 3 times in each group. After inoculation, the fruits were cultured in the dark at 28 °C and we added 10 mL of sterile water with sterilized cotton or filter paper to maintain humidity. The incidence of fruits and leaves was observed and recorded.
4. Discussion
Using fluorescent protein labeling to track the growth, development, and pathogenicity of fungal strains is an effective method in plant and animal pathogenic fungi. At the same time, fluorescent proteins are also widely used to study the spatial-temporal expression of genes, and the cellular and subcellular localization of proteins. At present, many different fluorescent proteins have been applied in fungi, such as
Neurospora crassa,
M. oryzae, and so on [
21]. However, compared with fungal species, such as
M. oryzae and
F. graminearum, there are fewer studies on the molecular mechanism of the
C. aenigma infection and the labeling of organelles with fluorescent proteins. In this study, the peroxisome and nucleus of
C. aenigma were successfully labeled by GFP and the red fluorescent proteins DsRED and mCherry.
The number of nuclei and the dynamic processes of nuclear division, movement, and degradation are some of the important contents of fungal developmental biology [
22].
In the process of the appressorium formation of
M. oryzae, there is a precise regulation of nuclear division and distribution to daughter cells. As a multinucleated fungus, the mechanism of nuclear division, separation, and regulation of
C. aenigma may be more complex. The formation and biochemical metabolism of peroxisome are essential to the infection of
M. oryzae and
B. cinerea [
23]. The fluorescent transformants obtained in this study have high abundance expression, high fluorescence brightness, and good dispersion, and can accurately trace the location, size, and movement of the nucleus and peroxisome in
C. aenigma, which provides a reference and source of material for studying the growth, development, pathogenic process, and molecular mechanism of the fungus.
The appropriate promoter to trigger the expression of the fluorescent proteins is the basis of fluorescent labeling. The results showed that H3 and MPG1 promoters from M. oryzae could be used to trigger the gene expression in C. aenigma. The fluorescent protein genes under the two promoters were abundantly expressed in the cells of C. aenigma in hyphae and conidia. The expression intensity was adequate for labeling the peroxisomes and the nuclei. Our results thus gave more options for effective fungal promoters regarding the study of fungal genes. In order to show the localization more clearly, we also combined applying the localization of fluorescent proteins with chemical fluorescent staining. We provided important data for the study of cell structure, gene function, and protein-protein interaction.
The transformation efficiency of C. aenigma is comparable to that of filamentous fungi, such as M. oryzae and F. graminearum, but the genetic stability of the transformants of C. aenigma is lower, which may be due to the multinucleation of C. aenigma. In addition, the colony morphology, growth rate, sporulation, and pathogenicity in most of the transformants were not significantly different from those of the wild type. The results indicated that the growth and development of most of the transformants were not affected by the transformation processes, or the expression of hygromycin and fluorescent proteins.
Therefore, most of the fluorescent strains would be capable of tracing the organelles in further cellular research. A very small portion of the transformants showed phenotypic variation, in which the T-DNA may have been inserted into some important gene loci. These transformants were important in identifying the functional genes in fungal molecular biology. Thus, our work provides a useful tool for the study of molecular and cellular biology in fungi.