Development of Transformation System of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: Hyphomycetes) Based on Nitrate Reductase Gene of Aspergillus nidulans

Abstract

A heterologous transformation system was developed for V. lecanii based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. Mutant no. 01 and 04 was chosen for the transformation experiments. Plasmid pBT was used as transformation vector containing the Aspergillus nidulans nitrate reductase gene. A frequency of approximately 3 transformants/μg DNA was obtained using the circular vector pBT. The establishment of a transformation system for V. lecanii is fundamental for genetic manipulation of this microorganism.

Introduction

Verticillium lecanii (Zimm.) Viegas, an important entomopathogenic fungus, acts as a promising biologic control agent for various insect pests. Verticillium section Prostrata was reclassified in the genera Lecanicillium, Pochonia, Haptocillium and Simplicillium based on morphological observations and molecular analysis [1–5]. The high virulence and epizootic efficiency of some Verticillium lecanii strains towards certain insects has been used to develop commercial mycoinsecticides like Mycotal and Vertalec for the biological control of aphids, whiteflies and other insects.

Investigation on this fungus covered the molecular basis of development and virulence has extended our knowledge of fungus-insect interaction mechanism in recent years [6]. Moreover, several genetic transformation systems [7] and gene knockout techniques have been developed in this species to facilitate the study of gene function in vivo. An effective gene expression system is required for molecular biology researches. Although high level of transgenes can be achieved by using constitutive promoters, constant expression of some genes may generate negative effects in transgenic tissues, especially inappropriate expression of genes is either highly deleterious or lethal [8]. Thus, a suitable expression system is required for temporal and spatial regulating gene expression.

Development of Verticillium spp. as biopesticides requires the development of a genetic technique by which desirable traits can be combined to produce superior strains. Strain improvement now embodies a more rational approach in respect to the strategies used for screening the mutated population of cells thus aiming at production of marker genes to enhance or remove specific activities, or increase virulence by selection of target hosts. Mutants with altered morphology, nutritional requirements, enzyme production, fungicide resistance and virulence have been isolated using chemical agents from entomopathogenic fungi, like Metarhizium anisopliae.

Successful transformation of fungi has been achieved by Calcium Chloride/Polyethylene glycol [9, 10], electroporation [11–13], particle bombardment [14, 15] and Agrobacterium tumifaciens mediated transformation (AMT) for development and analysis of a homologous or heterologous gene transfer system. The nitrate system is powerful and relevant in genetically poorly characterized organisms, especially pathogenic fungi, where other methods of mutant selection can introduce pleotropic and undesirable mutations. Therefore, a positive selection procedure is isolation of mutants lacking enzyme activity. Chlorate resistance allows the recovery of nitrate non- utilizing mutants in several fungal species such as Aspergillus nidulans, Fusarium oxysporum and provides an interesting system for the positive selection of mutation mapping at several different genes.

The most fertile source for molecular insights into transformation and gene manipulation strategies lies in the cell’s DNA. RAPD technique is a modification of PCR and allows to reveal polymorphism within completely unknown samples without the need of probe hybridization or DNA sequencing [16]. RAPD have been used for the characterization of V. dahliae isolates from different host species [17].

Thus, the aim of the present study was to develop a heterologous transformation system for V. lecanii based on the complementation of the nitrate reductase gene that would allow the genetic manipulation of this species.

Materials and Methods

Conidia of Verticillium lecanii parasitizing larvae of Culex quinquefasciatus were isolated from naturally infected mosquito larvae at Jabalpur region in the following manner:

Routine collection of water samples was made at regular intervals from mosquito breeding sites followed by isolation of entomopathogenic fungi from these natural waters on SMYA medium. Isolates were purified and working cultures were maintained as monosporic colonies on SMYA (which contained (in gl⁻¹): peptone 10; maltose 40; yeast extract 10 and agar–agar 20—pH 7.0) and incubated at 28 ± 1°C for 7–10 days.

Transformation Vector

Plasmid pBT was used as transformation vector. The 3.2 kb pBT bait plasmid carries a low copy p15A replication origin and contains Aspergillus nidulans nitrate reductase genes. It has multiple cloning sites and confers chloramphanicol resistance.

Isolation of Mutants of Verticillium lecanii

NiaD⁻ mutants of Verticillium lecanii were generated essentially according to a procedure developed by Cove [18]. Minimal chlorate medium (MMC) amended with 5% potassium chlorate was used to generate niaD⁻ mutants. MMC contains(l⁻¹ distilled water) : 30 g glucose, 1 g K₂HPO₄, 5 g MgSO₄·7H₂O, 5 g KCl, 0.01 g FeSO₄, 0.169 g Glutamate(10 mM), 50 g KClO₃, 20 g Agar–agar. Mycelial plugs were placed on the chlorate media either at the center or at 3–6 pts. on petriplates (9 cm diameter) and incubated at 28 ± 1°C. Chlorate resistant sectors, evident after 10–15 days, were transferred to minimal medium amended with NaNO₃, NaNO₂ and hypoxanthine respectively at 10 mM concentration used for partial phenotyping of chlorate resistant mutants. Mutants unable to utilize NO₃ as sole N₂ source were considered niaD⁻ mutants. Those which failed to utilize NaNO₂ as N₂ source were considered niiA⁻ mutants. Similarly those mutants which did not utilize hypoxanthine were cnx⁻ mutants.

Isolation of Protoplast

Protoplasts of Verticillium lecanii were isolated as per the following protocol. NiaD- mutants of Verticillium lecanii were grown in shake cultures of Sabouraud’s Dextrose broth for 3 days. Juvenile mycelium was harvested by filtering through muslin cloth, washed with digestion medium (1.2 M sorbitol and 10 mM Tris, pH 7), and then transferred to a flask to which 5 ml of digestion medium was added. To it, filtered BSA (12 mg BSA in 1 ml digestion medium) and Novozyme 234 (20 mg in 1.25 ml stabilizing medium [0.02 M PO₄ buffer pH 7.2 containing 0.6 M KCl and 2 mM MgCl₂] was added and the mixture was kept at 4°C for 15 min to start protoplast release from the mycelium. Subsequently, the mixture was incubated at 30°C in an orbital shaking incubator at 20 rpm.

The release of protoplasts from the mycelium of the mutant strain of the fungus was observed under low power microscope after every 30 min, until the mycelium was converted into protoplast. After 3 h, the flask was kept at 4°C for 15 min to stop the activity of Novozyme 234. The contents were filtered through muslin cloth and the filtrate was centrifuged at 2000×g for 7 min. The pellet was washed 2–3 times with 5 ml of digestion medium and again centrifuged for 5 min. The pellet was again washed with 5 ml of transformation medium(TM) {1.2 M Sorbitol; 10 mM Tris; pH 7.5 and 50 mM CaCl₂} and centrifuged at 40× for 5 min. The pellet was resuspended in 500μl of TM on ice and the yield checked by haemocytometer count.

Protoplast Regeneration

The fungal protoplasts suspended in transformation medium were centrifuged at 40×g for 5 min. Pellet was resuspended in 500 μl of TM on ice and the yield checked by haemocytometer count and then plated over regeneration agar which contained (in gl⁻¹) Sucrose 2; peptone 1; NaCl 0.5; yeast extract 3 and agar–agar 20 and incubated at 28°C for regeneration and to test the viability of protoplast. Regeneration frequency of protoplast was determined according to the following formula:

Fungal Transformation: By PEG (Polyethylene Glycol)

Protoplasts (2 × 10⁷) of niaD⁻ mutants of Verticillium lecanii in 200 μl were mixed with 15 μl of pBT plasmid and the mixture was incubated on ice for 30 min. In another set, protoplasts were mixed with pBT plasmid without niaD gene that was taken as control. To both the sets was added, 50 μl of 60% PEG 3000 (60% w/v in TM) and allowed to stand on ice for further 10 min. Then 1 ml of PEG was added to this and mixed by rolling the tube gently for few minutes and then inverted 3–4 times and incubated at room temperature for 10 min. The protoplast was diluted in 4 ml STC (1.2 M Sorbitol, 10 mM Tris(pH 7.5) and 10 mM CaCl₂) and centrifuged at 2500×g for 5 min. Pellet was resuspended in 400 μl of STC. Aliquots containing 2 × 10⁵ protoplasts were mixed and spread in petriplates with 30 ml of molten (45–48°C) regeneration medium which contained (1.2 M Sorbitol, 10 mM Tris pH 7.5, 0.1%K₂HPO₄, 0.05%MgSO₄, 3%NaNO₃ and 1.5%agar).

Mitotic Stability

Mitotic stability of the niaD⁻ transformants was examined by growth on a non-selective medium (complete medium). Cultures were sequentially transferred to nonselective medium five times and cultured 5 days each time. Cultures were then transferred back to selective medium (minimum medium with nitrate as sole nitrogen source) to determine whether the selective marker had been lost.

Analysis of Transformants by RAPD Analysis

The study was undertaken to identify RAPD markers for characterization of mutant, wild type and transformant of test strain of Verticillium lecanii. Although there are number of procedures for the isolation and characterization of DNA from fungi but modified method of DNA extraction has made possible to obtain DNA with extreme easiness from different host species [19]. Prior to other reactions, all DNA samples were denatured so as to activate harmful protease or nucleases in DNA samples. In a microfuge tube (0.5 ml), 100 ng of DNA sample was taken and denatured at 98°C for 5 min in a thermal cycler, after which the tube was kept at −20°C. Components of PCR were mixed in the following proportion to obtain a final volume of 20 μl-{Sterilized Distilled water-11.7 μl, 10× buffer-2.0 μl, MgCl₂ (3.5 mM)-2.8 μl, DNTPs (2.5 mM each)-1.5 μl, Primer (0.6 μM)-0.5 μl, DNA template (100 ng)-1.0 μl}

The random 10-mer primer selected for comparing and assessing the genetic difference between wild type, mutant and transformant of Verticillium lecanii was OPB-18 (5′CCACAGCAGT3′). DNA samples used as templates were dissolved in 10 Mm Tris HCl (pH 7.6) and 0.1 mM EDTA (pH 8). DNA was completely denatured by heating reaction mix at 94°C for 5 min. At the same temperature, 0.5 μl of thermostable Taq DNA Polymerase (in storage buffer-50% glycerol) was added. The reaction mix was overlaid with mineral oil (to prevent evaporation of samples). All reaction mixtures were prepared at 4°C and sealed before thermal cycling.

Reaction mix (in microfuge tubes) was kept in the thermoblock of PCR device. 16 well thermocycler was employed for thermocycling. After the reaction mix containing microfuge tubes have been loaded, the heated lid was closed over the thermoblock and locked. Repeated cycles of denaturation, annealing and extension were carried out in thermal cycler in the following manner:

PCR was performed in a series of about 39 repeated temperature changes called cycles; each cycle typically consists of three discrete temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage.

• Initialization step: This step consists of heating the reaction to a temperature of 94–96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1–9 min. It was only required for heat activation of DNA polymerases.

• Denaturation step: This step was the first regular cycling event and consisted of heating the reaction to 94°C for 5 min. It caused DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

• Annealing step: The reaction temperature was lowered to 40°C for 2 min allowing annealing of the primers to the single-stranded DNA template.

• Extension/elongation step: A temperature of 72°C was set with for about 2 min. At this step the DNA polymerase synthesized a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′–3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand.

• Final elongation: This single step is occasionally performed at a temperature of 72°C for 10 min after the last PCR cycle to ensure that any remaining single-stranded DNA was fully extended.

All samples were taken out after the reaction cycling was over. Mineral oil was removed from the samples by extraction with 150 μl chloroform. Amplification reaction products appear as a micelle near the meniscus in the aqueous phase. This micelle was transferred to a fresh microfuge tube. DNA Amplification reaction products appear as a micelle near the meniscus in the aqueous phase. This micelle was transferred to a fresh microfuge tube. DNA Amplification reaction products were mixed with 2.5 μl of 10× gel loading dye buffer (0.25% bromophenol blue, 0.25% xylene cyanol and 40% sucrose w/v) and microfuged before loading. Amplification reaction products were analysed by electrophoresis in 1.2% agarose gel using TBE buffer with Ethidium Bromide at 130 V/cm. Size of amplified fragments was estimated using 100 bp DNA ladder as a molecular weight marker.

Results & Discussion

Selection of Nitrate Reductase Mutants

After 10 days of growth at 25°C, five Verticillium lecanii chlorate resistant mutants were selected (Table 1). Mutants with the chlorate resistance phenotype can be obtained by mutation in different genes [18] like the nitrate permease gene (crnA), and in the proper nitrate reductase (niaD) gene. Independent of which gene is affected, mutation will prevent the cell from reducing chlorate to chlorite. These mutations can be easily differentiated on the basis of growth tests.

Table 1.

Growth of Verticillium lecanii chlorate mutants in minimal medium containing different nitrogen sources

Mutation	Number of Mutants	Nitrogen source
		Nitrate	Nitrite	Glutamate	Ammonium
niaD	02	−	+	+	+
niiA	01	−	+/−	+	+
cnxA	02	−	−	+	+
Total	05

All nitrogen sources were adicioned in a concentration of 10 mM. + indicates normal growth, and − indicates absence of growth

Nitrate reductase mutants were obtained in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. Since the isolated mutants were spontaneous and because mutation is a random event, the number of isolated niaD⁻ mutants for each species will predictably not follow a pattern, as reported for Paecilomyces griseoroseum, where 8.33% of the chlorate resistants mutants were niaD⁻ mutants, while in P. canescens the isolated niaD⁻ mutants made up 49.0% [20].

After the isolation of niaD⁻ mutants, the recipient strain for transformation procedures must be chosen. All the 05 niaD⁻ mutants were tested for possible revertents and the reversion frequency was <1 × 10⁻⁶ in all of them. The mutant no. 01 and 04 were chosen for the transformation experiments since they presented the lowest residual growth in minimum medium containing nitrate as sole nitrogen source.

Transformation of Protoplasts

Successful application of gene cloning technology to these entomopathogenic fungi relies on an efficient transformation system. Therefore, in the present investigation, protoplasts were successfully isolated from the juvenile mycelium of V. lecanii (niaD⁻ mutant) by incubation with Novozyme 234 for 4 h. These protoplasts regenerated at a frequency of 39.84–41.40% (Table 2).

Table 2.

Virulence and cultural characteristics of wild type and transformant strains of Verticillium lecanii

S. no.	Strain	LT50	Radial growth rate (mm/day)	Germination
1.	V.lecanii- wild type	8.4 ± 0.1	1.6 ± 0.4	37 ± 4
2.	V.lecanii- transformant 1	6.1 ± 0.3	1.3 ± 0.7	35 ± 6
3.	V.lecanii- transformant 2	5.8 ± 0.6	1.02 ± 0.8	36 ± 3

Transformation of protoplasts is carried out by conventional PEG method. The transformed protoplasts grew in presence of nitrate in minimal medium supplemented with chlorate whereas the untransformed protoplasts failed to grow in presence of nitrate. Transformation of V. lecanii with plasmid pBT resulted 39 transformants per μg DNA with niaD. Of them, mitotically stable transformants were 17 in number, whereas 22 transformants were found to be unstable. Fang [19] transformed B. bassiana with binary plasmid pBANF-bar-Bchit1. Sandhu et al. [21] developed a heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae based on the use of A. nidulans nitrate reductase gene (niaD). The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8–20 transformants perμg of DNA.

Mitotic Stability of Transformants

Upon analysis of the genetic stability of the transformants, it was found that out of 39 transformants analyzed with PEG method, 43.5% were genetically stable. Bogo et al. [22] reported comparatively low mitotic stability of 46% among 44 transformants analyzed by PEG method in M. anisopliae.

Virulence of Transformants

The virulence of wild type strain of V. lecanii and that of transformants towards larvae of C. quinquefasciatus were analyzed and it was concluded that wild type strain of V. lecanii was less virulent than the transformant (Table 2). Also, spores extracted from insect cadavers inoculated with transformants germinated on SMYA at a faster rate than spores of wild type V. lecanii. The relative increase in virulence among the transformants, was associated with faster rate of spore germination. Radial growth rate in vitro was high among the transformants. Similar findings were reported by Goettel et al. [23] who transformed the insect pathogenic hyphomycete, Metarhizium.

Analysis of Transformants by RAPD Analysis

The comparative strains were characterized using PCR-RAPD analysis. The DNA yields from transformant, mutant and wild type strain of V.lecanii were reasonably high, and a clear band pattern was frequently seen when 150 ng DNA preparation was run on 0.8% agarose gel at 80 V/cm for 1 h, stained with ethidium bromide and photographed using UV transilluminator. The material analysed comprised of three accessions, namely DNA of mutant strain, DNA of wild type strain and of the transformant of V. lecanii. The sizes of amplified DNA fragments varied from 80 to 200 bp and a total of 5 bands were scored from the selected random primer of operon technology (USA). The DNA bands were recorded on the basis of their presence or absence in the organism. The results shown in Fig. 1 indicates that there were significant differences between the RAPD pattern. It is clearly revealed from the RAPD electrophoretic profile that a band of 200 bp is seen in lane 3 which is genomic DNA of wild type strain of V.lecanii and also in lane 2, DNA of the transformant whereas in lane 1, DNA of mutant strain, this particular band is not present. Hence, the presence of transformant was evident by the appearance of the extra band. OPB-18 (5′CCACAGCAGT3′) was the 10-mer primer used in the present investigation. As this 10-mer primer is not specific probe, multiple flanking regions also had amplified bands of 150–50 bp which confirms the species level is common. Thus, the result confirms the successful transformation of niaD gene in the mutant strain. RAPD-PCR analysis has been used to differentiate Verticillium lecanii strains isolated from insect (Homoptera) and from rust fungi for analysis of virulence against whiteflies [24].

Fig. 1.

RAPD-PCR banding pattern showing comparative analysis of wild type, mutant and transformant (V. lecanii)

Modern molecular biology techniques offer powerful tools for analyzing interactions between organisms. Use of pBT plasmid as transformation vector is useful in developing a heterologous transformation system for entomopathogenic fungi based on the complementation of the nitrate reductase gene that would allow the genetic manipulation of this species [25, 26]. With increasing understanding of biological phenomena, efficiency of biocontrol agents could be improved by transferring useful genes between micro-organisms.

Hence, in the present investigation, attempts are made to breed the strain of V. lecanii using several important parameters associated with expression of virulence, to act as an improved biocontrol agent by combining markers for virulence to mosquitoes employing protoplasts and transformation system strategies. This will be a part of long term isolate improvement program for this biological control agent.