Comparison of different protein extraction methods for gel-based proteomic analysis of Ganoderma spp.

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant–fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp

Comparative proteomic analysis of Ganoderma species during in vitro interaction with oil palm root

Basal Stem Rot (BSR) in oil palm caused by Ganoderma spp. is a deadly disease affecting oil palm plantation yield and global cooking oil supply. A pathogenic species, Ganoderma boninense is claimed as the main causal agent of BSR while Ganoderma tornatum is regarded as non-pathogenic and unable to infect living palms. Insufficient information on the infection mechanism and immature early detection strategy of the pathogen are among the disease control limitations. The existing molecular studies on the oil palm-Ganoderma interaction mainly focused on the response of the plant towards the fungus infection while the information on the pathogen responses is still a scarce. Therefore, in this study, response of the fungus at the infective stage during interaction with oil palm at the molecular level was investigated. An optimized protein extraction protocol for 2-Dimensional Electrophoresis (2-DE) gel analysis of Ganoderma spp. was developed and a comparative proteomic analysis were conducted to investigate the changes in the dikaryotic mycelial protein expression of the pathogenic G. boninense and non-pathogenic G. tornatum during in vitro interaction with oil palm root. The phenol/ammonium acetate in methanol was shown to be the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp. mycelia. Scanning Electron Microscope (SEM) images obtained confirmed the hyphae attachment and colonisation of both species on the oil palm root surface after 72 h of inoculation. Comparative proteomic analysis showed that the mycelial proteins from oil palm root exhibited different expression profiles when compared to the mycelia grown on Potato Dextrose Agar (PDA). Proteins differentially expressed in both species may have either direct or indirect link to virulence and pathogenicity, metabolism, growth and maintenance of both Ganoderma species. During the interaction, proteins with potential contribution to fungal pathogenicity such as enolase, alpha-aminoadipate reductase, carboxypeptidase, dienelactone hydrolase, glutamine synthetase (GS) and guanine nucleotide binding proteins (G proteins) were upregulated in G. boninense while bZIP protein, triose-phosphate-isomerase, redoxin and peroxiredoxin were mutually expressed in both species. Identification of these proteins during the interaction with oil palm roots may provide fundamental information for further investigation on specific roles of the identified proteins towards Ganoderma infection mechanism and facilitate selection of potential markers for early detection of BSR in the future

Comparative proteomic analysis of Ganoderma species during in vitro interaction with oil palm root

Basal stem rot disease caused by Ganoderma spp. is still considered a large threat to oil palm production in many countries, especially in Malaysia, which contributes to approximately 45% of the world's palm oil exports. In the last 10 years, studies on the oil palm-Ganoderma interaction have mainly focused on the response of the plant towards the fungus. In this study, a comparative proteomic analysis was conducted to investigate the change in protein expression of two Ganoderma spp., namely Ganoderma boninense, which is known as the most aggressive species and Ganoderma tornatum, previously reported as non-pathogenic. Our results showed that both species colonize and penetrate the oil palm root after only 72 h of inoculation. In addition, proteins were expressed differentially in both species that have either direct or indirect links to virulence and pathogenicity. Other proteins related to fungal growth, metabolism and stress from both species were also discussed in this study

Molecular investigation of carrageenan production in Kappaphycus alvarezii in different culture conditions: a proteomic approach

Carrageenan is a polysaccharide extracted from different species of red seaweed for use in pharmaceuticals and cosmetics industries. In Malaysia, κ-carrageenan is obtained through extraction from the tropical red seaweed Kappaphycus alvarezii. The use of tissue culture techniques in the propagation of K. alvarezii has proven to be effective in solving cultivation problems and produced high-quality seedlings and providing a sustainable source of better quality carrageenan. In order to understand the molecular mechanisms behind that, a proteomic investigation was conducted comparing the changes in protein expression in tissue-cultured and liquid-cultured K. alvarezii after 60 days of cultivation. Proteomic analysis was used to study the changes in the protein expression level between liquid-cultured and tissue-cultured of the red seaweed K. alvarezii. A total of 45 protein spots were found to be significantly different in their densities and three proteins, namely β-amylase, NAD-dependent sugar epimerase and B-phycoerythrin, showed a consistent pattern of upregulation in ELISA analyses, hence validating the 2-DE profiles. Changes in the proteins expression level were noticed in proteins related to energy production, metabolism and cellular maintenance. The protein changes in tissue-cultured seaweed possibly play an essential role in the production of carrageenan