Molecular defense response of oil palm to Ganoderma infection.

Phytochemistry xxx (2014) xxx–xxx

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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Review

Molecular defense response of oil palm to Ganoderma infection C.-L. Ho a,b,⇑, Y.-C. Tan b,1 a b

Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM-Serdang, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM-Serdang, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Ganoderma Oil palm Elaeis guineensis Jacq. Basal stem rot Molecular defence Lignin

a b s t r a c t Basal stem rot (BSR) of oil palm roots is due to the invasion of fungal mycelia of Ganoderma species which spreads to the bole of the stem. In addition to root contact, BSR can also spread by airborne basidiospores. These fungi are able to break down cell wall components including lignin. BSR not only decreases oil yield, it also causes the stands to collapse thus causing severe economic loss to the oil palm industry. The transmission and mode of action of Ganoderma, its interactions with oil palm as a hemibiotroph, and the molecular defence responses of oil palm to the infection of Ganoderma boninense in BSR are reviewed, based on the transcript profiles of infected oil palms. The knowledge gaps that need to be filled in oil palm–Ganoderma molecular interactions i.e. the associations of hypersensitive reaction (HR)-induced cell death and reactive oxygen species (ROS) kinetics to the susceptibility of oil palm to Ganoderma spp., the interactions of phytohormones (salicylate, jasmonate and ethylene) at early and late stages of BSR, and cell wall strengthening through increased production of guaiacyl (G)-type lignin, are also discussed. Ó 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ganoderma spp. reported in BSR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Life cycle of Ganoderma spp. and its relation to BSR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Transmission of BSR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Development and progress of BSR in oil palm roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant-pathogenic interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene expression of oil palm roots in response to Ganoderma infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Oil palm (Elaeis guineensis Jacq.) is one of the main sources of edible oil in the world with Indonesia and Malaysia contributing about 85% of the world’s palm oil production. Total export revenue

⇑ Corresponding author at: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPMSerdang, Selangor, Malaysia. Tel.: +60 3 89467475; fax: +60 3 89467510. E-mail address: [email protected] (C.-L. Ho). 1 Present address: Codon Genomics Sdn. Bhd., No. 11/A-2, Jalan Bandar Lapan Belas, Pusat Bandar Puchong, 47160 Puchong, Selangor, Malaysia.

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of oil palm products (palm oil, palm kernel oil, palm kernel cake, oleo chemicals, biodiesel and finished products) for Malaysia and Indonesia in 2012 was approximately USD 22 billion (bepi.mpob.gov.my/; www.indonesia-investments.com/), respectively. In order to meet the high demand for palm oil which increases annually, the oil palm plantation areas in these two main palm oil producing countries in South East Asia have expanded. Over the last three decades, the oil palm plantation area in Malaysia has expanded from 641,791 hectares in 1975 to 5.23 million hectares in 2013 (bepi.mpob.gov.my), while the total oil palm plantation area in Indonesia has reached 8 million hectares in 2010 (Rianto et al., 2011). Meanwhile, breeding and selection of palms with high yield

http://dx.doi.org/10.1016/j.phytochem.2014.10.016 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ho, C.-L., Tan, Y.-C. Molecular defense response of oil palm to Ganoderma infection. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.016

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C.-L. Ho, Y.-C. Tan / Phytochemistry xxx (2014) xxx–xxx

potentials and disease tolerance (thus reducing yield loss due to diseased palms) are crucial for sustainable production of palm oil without further expansion of plantation area. Hence, an understanding of oil palm disease infection process and host defence mechanisms at the molecular level are required. Oil palm is susceptible to a number of fungal diseases, including Phytophthora, Fusarium wilt, upper stem rot (USR) and basal stem rot (BSR) (Turner and Bull, 1967; Aderungboye, 1977). Among these, USR and BSR are caused by pathogenic fungi belonging to the genus Ganoderma and BSR is considered to be one of the most devastating oil palm diseases. BSR is an oil palm root disease which manifests itself as decay of roots and lower stem, and can be spread by direct root contact and basidiospores by still unknown means (Pilotti et al., 2002; Rees et al., 2007, 2009, 2012). Ganoderma spp. are white rot fungi that are capable of degrading components of the plant cell wall including lignin (Paterson, 2007). The fungal pathogen disrupts water and nutrient transport to the upper part of the palm thus causing frond wilting, yellowing of fronds, unopened spear leafs, reduced and ‘‘one-sided mottling’’ of canopy, and emergence of basidiocarps on the lower stem (Turner, 1981; Chung, 2011). Infected oil palms were reported to have a lower oil yield before the stands collapsed eventually (Singh, 1991). To make the condition worse, BSR which was initially thought to infect only mature palms over 25 years of age (Thompson, 1931), was found to infect also young palms (10–15 years) (Turner, 1981) and seedlings less than 5 years old (Singh, 1991). In this review, the focus is mainly on BSR since it is more prevalent in most of the oil palm plantations in South East Asia. BSR occurs frequently in coastal marine clay areas which were previously planted with coconut trees in western Peninsular Malaysia (Navaratnam, 1964; Lim et al., 1992). However, it was also reported to happen in peat soil situated inland (Ariffin et al., 1989; Rao et al., 2003). In order to control and suppress the spread of the disease, several approaches were explored. Practices that involved fungicides and burning of infected oil palms are unfavorable to the environment. Furthermore, most fungicides are not effective or specific against BSR (Idris and Arifurrahman, 2008). Meanwhile, conventional cultural practices, such as elimination of infected palms and improved sanitation processes, can only delay the spread of the disease (Breton et al., 2006), while extensive physical clearing of infected palms is not economically feasible. Biological controls of BSR could be the most suitable approach, as it is safe for the users, and poses little threat to non-target organisms. However, to develop a highly selective biological control for BSR is difficult, expensive, time consuming, and less effective when applied in the field (Damon, 2000). Host resistance can be used in preventing and controlling plant diseases. Oil palms from different genetic origins with differences in disease susceptibility to BSR have been reported (Idris et al., 2004; Durand-Gasselin et al., 2005). They could serve as useful genetic resources for the improvement of oil palm tolerance or resistance to Ganoderma spp., employing a similar strategy which was used to improve the resistance of oil palm to Fusarium wilt (Cochard et al., 2005).

2. Ganoderma spp. reported in BSR The fruiting body of Ganoderma lucidum has gained wide popularity as a dietary supplement especially in China, Taiwan and, Japan for its perceived health benefits in preventing immunological diseases, such as hypertension and tumorigenesis (Liu et al., 2002). Many Ganoderma species are also plant pathogens to khair, grapevines, betel palm, rubber, tea, and oil palm (Turner, 1965; Bakshi et al., 1976; Adaskaveg and Gilbertson, 1987). G. lucidum was first reported as a causal agent of BSR in oil palm in 1930 (Thompson,

1931; Utomo et al., 2005). Subsequently, six additional species were reported to be associated with BSR in Malaysia and Indonesia, including Ganoderma boninense, Ganoderma tornatum, Ganoderma chalceum, Ganoderma zonatum, and Ganoderma xylonoides (Steyaert, 1967). To date, a total of 15 Ganoderma species have been detected in oil palms (Turner, 1981). Although G. boninense was identified as the most virulent species that caused BSR, multiple species could be responsible for the disease on individual trees (Ho and Nawawi, 1985). 2.1. Life cycle of Ganoderma spp. and its relation to BSR Ganoderma spp. are classified as basidiomycetes. The life cycle of G. boninense in relation to BSR has been studied by Hasan and Flood (2003). Generally, each basidiospore germinates into a genetically unique monokaryotic hypha which is saprophytic and able to colonize dead palm wood. Ganoderma spp. have a tetrapolar mating system which favors outcrossing. They are heterothallic with two pairs of alleles at two mating loci, thus ensuring maximum genetic diversity by restricting inbreeding to 25% (Rees et al., 2009). Hyphae of compatible mating type anastomose to produce a dikaryotic mycelium which could be potentially invasive. This heterokaryon proliferates and grows, with two haploid nuclei dividing and multiplying independently in each septated unit until the life cycle of Ganoderma is completed when the dikaryotic hyphae produce a fruiting body known as a basidiocarp (Hasan and Flood, 2003). The basidiocarp bears specialized cells called basidia which resemble little clubs where karyogamy occurs. The basidia then divide meiotically to produce genetically unique basidiospores (Campbell et al., 2008). 2.2. Transmission of BSR Various sources of infection and modes of transmission, have been proposed for BSR. Basidiospores have been proposed as a source of BSR infection (Pilotti et al., 2003; Rees et al., 2012). Basidiocarps are able to release a high number of basidiospores which can travel a long distance (Sanderson, 2005) and become a source of infection for a wounded palm surface created during plantation harvesting and management (Rees et al., 2012). Anastomosis of basidiospore germlings could occur on palm surface, debris or fallen palms in soil causing direct infection via cut fronds or indirect infection through roots (Rees et al., 2012). Spread through basidiospores must be accountable for from the genetic diversity of Ganoderma isolates from the field (Miller et al., 1999; Pilotti et al., 2003). Root invasion was considered as the primary route of transmission of BSR (Rees et al., 2009). Since Ganoderma spp. have poor competitive saprophytic capability in soil, colonized debris left in the field by infested palms from previous planting of coconut or oil palm have been proposed as a very important source that provides the substantial amount of inocula required for root infection (Hasan and Turner, 1998; Rees et al., 2007). The infection of oil palm is likely to occur when the roots come into contact with inocula from the debris left in the ground (Flood et al., 2005), or from roots of neighboring infected palms. The tetrapolar heterothallism of Ganoderma spp. also explains a number of phenomena: 1. Genetic diversity of isolates within a plantation was as great as between plantations whereby the vast genetic diversity of fungi observed on the infected palms could be a result of plasmogamy of genetically different mycelia derived from basidiospores; 2. BSR takes a long time to be evident in the field due to the requirement of plasmogamy or anastomosis of compatible



mating types to form virulent dikaryotic mycelium, and they have weak competitive ability in soil or on organic debris (Rees et al., 2007); 3. BSR symptoms may not be detectable even though Ganoderma spp. can be detected on palms whereby the fungus could be in the form of monokaryotic mycelia living saprophytically on dead tissues on the palm surface since monokaryotic mycelium are non-infective (Goh, 2005; Rees et al., 2007). 2.3. Development and progress of BSR in oil palm roots The development and progress of BSR in oil palm roots have been studied by Rees et al. (2009) in great detail. Briefly, infection by Ganoderma spp. is initiated by the penetration of oil palm root surface (epidermis and exodermis) by fungal mycelia, followed by a longitudinal progression of hyphae through inner and thin-walled cortex, and colonization of the lower stem (bole) eventually. Host cells in newly colonized tissue were shown to be colonized by intracellular hyphae and contained intact cell wall, intact cytoplasm, and organelles (Rees et al., 2009). At this stage, depletion of starch grains in the cytoplasm was observed in host cells in advance of invasion and in the lower stem of infected oil palm (Rees et al., 2009). Ganoderma spp. may behave as hemi-biotrophs in newly colonized tissues before turning into necrotrophic pathogens as implicated by extensive degradation of host cell walls. Cell wall degrading enzymes (CWDEs) such as cellulase, manganese peroxidases and laccases that are involved in the degradation of cellulose and lignins, are expected to be released by the fungus. At this stage, the host cells were colonized by hyphae intra, intercellularly and intramurally. Rees et al. (2009) also suggested that the defense response of oil palm may rely on production and release of antimicrobial compounds rather than on cell wall strengthening. In the subsequent stage, the oil palm roots are surrounded by a tough and melanised mycelium (pseudo-sclerotium) with thin-walled hyphae encased by many thick-walled cells (Rees et al., 2009). This leads to massive hyphal aggregations outside the oil palm roots, culminating in the formation of basidiocarps and release of basidiospores (Rees et al., 2009). An infected oil palm tree may or may not bear any fruiting bodies. The presence of a fruiting body on an infected palm normally shows that the fungus has been in the wood for at least several years, and an extensive decay could have taken place in the stem (Najmie et al., 2011). 3. Plant-pathogenic interactions The genetics of a host plant and its pathogen were first explained by the gene-for-gene hypothesis developed by Flor (1942). According to this hypothesis, the inheritance of both resistance in the host plant and ability of pathogen to cause disease is controlled by a pair of matching genes, namely the resistance (R) gene in the plant host, and the avirulence (Avr) gene in the pathogen. When a pathogen carrying a single dominant Avr gene is recognized by a single dominant R gene from a plant host, it fails to cause any disease. It is thus called an avirulent pathogen and the host is classified to be resistant, with their interaction known as being an incompatible interaction (Prell and Day, 2001). In the absence of an Avr gene in the pathogen and/or an R gene in the host, the pathogen is considered to be virulent and the host is susceptible, with their interaction known as a compatible interaction. In the latter, the pathogen is able to form a parasitic relationship with the host plant. R gene products may not bind to Avr gene products directly, but rather detect alterations in host proteins that are caused by the gene products from the pathogens, as explained by the guard model (van der Biezen and Jones, 1998). Other models

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for R activation including the switch model (Takken et al., 2006) and decoy model (van der Hoorn and Kamoun, 2008) have been described. The interactions of plants and microbial pathogens are among the most complex phenomena in nature, in which an unlimited variety of pathogenic molecules can interact with the cellular component of host plants (Schneider and Collmer, 2010). In brief, common microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) from microbes including non-pathogens such as flagellin, lipopolysaccharide, fungal chitin and b-glucans are detected by host plants (Nurnberger et al., 2004; Zipfel and Felix, 2005). Some pathogens may secrete CWDEs that can degrade host cell wall producing fragments of structural polysaccharides known as damage associated molecular pattern (DAMPs). The recognition of these patterns by transmembrane pattern recognition receptors (PRRs) activates the primary defense response in host plants (PAMP-triggered immunity, PTI) through signal transduction of a few signalling pathways (de Wit, 2007). PTI could negatively affect colonization of the pathogens through production of reactive oxygen species (ROS) and phytoalexins, cell wall alterations, deposition of callose, and accumulation of defence-related or pathogenesis-related (PR) proteins (van Loon et al., 2006; de Wit, 2007). In addition to PTI, plants have a secondary defense system (effector-triggered immunity, ETI) whereby the plant resistance proteins (RPs) encoded by R genes often with characteristic nucleotide binding (NB) and leucine rich repeats (LRRs), could monitor the presence of effectors or their perturbations and trigger RPmediated secondary responses. ETI and PTI respond to different pathogen-derived molecules and they differ by the intensity of their immune responses. The recognition of effectors can be direct (Dodds et al., 2006), or indirect (van der Biezen and Jones, 1998). In order to avoid recognition by PRP, biotrophic pathogens may produce multiple effectors that suppress PTI. The ETI induced by a biotrophic pathogen often results in a hypersensitive response (HR) and other locally induced defence responses that block further invasion of the pathogens. HR-associated cell death which confines pathogens at the infection site and limits their growth and supply of nutrients, is a typical indicator of host resistance to biotrophic pathogen. Over time, pathogens may evolve under selection pressure and acquire ability to overcome ETI and evade defense responses of host plants through complete removal or subtle changes of amino acids of effectors (Chisholm et al., 2006; Jones and Dangl, 2006). The functions of effectors include suppression of PTI; suppression of HR (Janjusevic et al., 2006); induction of abscisic acid (ABA) pathway to facilitate disease (de Torres-Zabala et al., 2007); production of metabolites that suppress salicylic acid (SA)- and jasmonic acid (JA)-induced defence responses; as well as establishment of disease symptoms (Zhao et al., 2003; Brooks et al., 2005). When an effector can no longer be detected by the host plants, fewer RPs will be induced. The failure of host plants to trigger ETI leads to susceptibility to pathogen. The presence of R gene or RP which is specific against BSR in oil palm has not been investigated. However, sequencing of a partiallength disease resistance gene homologue encoding NBS–LRR type R protein in oil palm has been reported (Chin et al., 2012). Many sequences related to R genes have also been recently identified from the transcriptomes of oil palm (Low et al., 2014). Despite this, the existence and involvement of the R gene in BSR which is caused by a hemi-biotroph, remain a speculation without evidence showing that these putative R proteins are involved in disease resistance. So far the R genes associated with plant resistance to necrotrophs have not been reported, except for a Toll/interleukin 1 receptor domain R protein in Arabidopsis which conferred resistance to Leptosphaeria maculans (Staal et al., 2008).


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The heterogeneity of commercial oil palm populations derived from crosses of dura and pisifera could create a selection pressure for Ganoderma spp. that favor outcrossing. If the R protein for BSR does exist, tetrapolar heterothallism may allow Ganoderma spp. to segregate for more aggressive pathogenicity to overcome host defense response. Given the way Ganoderma spp. reproduce and the genetic divergence of their basidiospores, DurandGasselin et al. (2005) have suggested breeding for partial resistance as a means for durable and long term solution to the BSR problem in oil palm. This can be achieved by integrating many genes of minor effect to provide reasonable levels of resistance in some oil palm lines. Furthermore, oil palm with an ‘‘all-or-nothing type’’ of resistance has not been reported. Biotrophs and necrotrophs are fundamentally different in their infection processes, the nature of effector proteins, and the elicited host-defence responses (Laluk and Mengiste, 2010). Biotrophs grow between host cells and invade relatively few host cells, as well as secrete a limited amount of CWDEs and generally lack phytotoxic compounds (Mendgen and Hahn, 2002). Through this feeding activity, biotrophs establish a long-term feeding relationship with the living cells of their hosts. Attempted infection by biotrophic pathogens would trigger plant immune responses, including HR which causes cell death at the site of infection. HR confines the spread of a pathogen by abolishing its nutrient supply, thereby limiting the growth of biotrophic pathogen. Conversely, cell death is an indicator of successful infection of necrotrophs which extract nutrients from dead cells (Govrin and Levine, 2000). In contrast to biotrophs, nectrophs secrete diverse phytotoxic compounds and CWDEs to induce cell necrosis and cause leakage of nutrients. Plant mutants with enhanced cell death showed increased resistance to biotrophic pathogens but were susceptible to necrotrophs (Veronese et al., 2004). Generally, cell death promotes susceptibility of plant host to necrotrophs; however, it is unknown whether it also applies to all necrotrophs (Laluk and Mengiste, 2010). Many fungi that are considered as necrotrophs may be hemibiotrophs, as they have biotrophic stage early in the infection process, including Ganoderma spp. are amongst this group (Rees et al., 2009). Infected oil palm may respond to the invasion of Ganoderma spp. (which behave as biotrophs) at the initial stage, by inducing HR. The induction of HR, which is in favor of necrotroph growth may trigger the switch of Ganoderma spp. from biotrophs to necrotrophs, and render the oil palm susceptible to BSR. Nevertheless, HR was reported to be associated with the resistance of plant hosts to two hemi-biotrophs Magnaporthe oryzae (Jia et al., 2000) and Phytophthora infestans (Vleeshouwers et al., 2000). In addition, ROS was found to have contrasting roles in plant defense depending on the lifestyle of the pathogens and the kinetics of the oxidative burst (Lamb and Dixon, 1997). At early stages of infection, ROS may act as signalling molecules that activate various immune responses in plants, resulting in resistance to both biotrophs and necrotrophs. Upon the establishment of infection, ROS may promote cell death causing HR-associated cell death in biotrophs, and also susceptibility to necrotrophs. The associations of HRassociated cell death and the kinetics of the oxidative burst to the susceptibility of oil palm to Ganoderma spp. await further investigation. Knowledge on the ability of oil palm cells to attenuate necrosis or to alleviate the effects of necrosis is lacking. The interplay of hormones such as JA, SA and ethylene and their related pathways also contribute to the resistance and susceptibility of plants to pathogens. JA signalling and ethylene-response pathway are important to the resistance of plant hosts to necrotrophic pathogens (Glazebrook, 2005; Laluk et al., 2011), whereas the accumulation of SA increases the resistance of plant host to hemibiotrophic pathogens but promotes the susceptibility to necrotrophic pathogens (Veronese et al., 2004, 2006). The roles of

different phytohormones during BSR have not been investigated. The question as to whether oil palm can also switch its defense responses rapidly through the interplay of hormones according to the switch in the feeding modes or life styles of Ganoderma spp. remains unanswered. The ability of necrotrophs to induce necrosis is central to their successful invasion, while the ability of host plants to counter fungal toxins (or other virulence factors) or their effects on necrosis are major factors in host resistance (Mengiste, 2012). Although the production of necrosis and ethylene inducing proteins which are able to cause cell death in dicotyledonous plants has been reported in necrotrophic fungal and bacterial species (Staats et al., 2007), little is known about production of phytotoxic compounds in Ganoderma spp., including their targets and ability to cause cell death to monotyledonous plants. The published genome of G. lucidum (Liu et al., 2012) may provide insights into the production of phytotoxic compounds and wood degradation by fungi belonging to the same genus. Although there are efforts by the Malaysian Palm Oil Board (MPOB; www.mpob.gov.my/Ganoderma/), ACGT Sdn. Bhd. (www.acgt.asia/press/pdf/02Nov2010.pdf), Malaysian Genomics Resource Centre (MGRC, (www.mgrc. com.my/)) and Felda Agricultural Services Sdn. Bhd. to sequence the genome of G. boninense in recent years, the sequences have not been made available in the public domain. The availability of this data will enhance our understanding of the potential virulence genes involved in Ganoderma infection of oil palm and enable us to develop better methods for BSR detection and to devise more effective strategies to prevent BSR. As white rot fungi, Ganoderma spp. are able to degrade the lignin component in host cell walls through the production and secretion of CWDEs such as lignin peroxidases, manganese peroxidases and laccases (Paterson et al., 2009; Liu et al., 2002). In general, lignin protects cell wall polysaccharides from microbial degradation. The lignin fraction in the oil palm trunk predominantly comprised syringyl unit (S-type lignin) (Sun et al., 1999), which is presumably more susceptible to degradation than the guaiacyl unit (G-type lignin). Paterson et al. (2009) have proposed improvement of oil palm resistance to Ganoderma spp. through genetic manipulation of lignin biosynthesis in oil palm. Technically, this can be achieved by producing transgenic oil palms with lignin which are rich in guaiacyl unit through genetic manipulation of enzymes/pathway involved in its biosynthesis. The enzymes and pathway involved in lignin biosynthesis have been well characterized in higher plants (Vanholme et al., 2010). The newly available oil palm genome (Singh et al., 2013) can be referred for relevant gene sequences to be transformed, and using well established oil palm transformation methods (Parveez et al., 2000; Abdullah et al., 2005), genetic engineering of oil palm for disease resistance can be applied. Overexpression of transcripts encoding key enzymes that produce the precursors of lignin units in the phenylpropanoid pathway, and polymerization of lignin, together with the suppression of enzyme that produce the S-type lignin such as coniferaldehyde (ferulate) 5-hydroxylase by the RNAi approach, could be attempted for this purpose. Nonetheless, the manipulation of enzymes involved in phenylpropanoid pathway may affect other related pathways such as the biosynthesis of flavonones/ flavonols which share the same precursors i.e. p-coumaric, cinnamic and caffeic acids. The partition of these precursors to different pathways justifies further research because the production of other important secondary metabolites such as phytoalexin isoflavonoids, which are important anti-microbial compounds, could be reduced at the expense of increasing lignin biosynthesis. Furthermore, Skyba et al. (2013) have shown that transgenic poplars with syringyl-rich lignin are more resistant to degradation by wood decay fungi including white rot fungi, suggesting that elevated guaiacyl content does not necessary improved decay



resistance of wood. The biochemistry of lignolytic enzymes from Ganoderma spp. should be investigated to shed light on the types of lignin monomers preferred by these enzymes. Paterson et al. (2009) also suggested the assessment of the lignin content of oil palm genetically transformed with Bacillus thuringiensis toxin genes and their resistance to Ganoderma spp. based on reports that corns transformed with B. thuringiensis toxin genes have increased lignin contents (Saxena and Stotzky, 2001; Poerschmann et al., 2005). However, Bt-corns were not found to have a significant higher content of lignin by Jung and Sheaffer (2004) and Poerschmann et al. (2008). Furthermore, the increase in total lignin in these plants could be due to random and unintentional insertion of B. thuringiensis toxin gene into genes that control lignin biosynthesis as explained by Paterson et al. (2009). If this is the case, it is unlikely that the oil palm genetically transformed with B. thuringiensis toxin genes will have a higher content of lignin. In regard to transgenic oil palms, the acceptability of palm oil from transgenic oil palms by consumers especially from the European market remains the main concern of palm oil producers and oil palm planters (Paterson et al., 2009), and should not be taken lightly. In light of that, screening and breeding for oil palm with cell walls enriched in the presumably more resistant G-type lignin could be performed despite its lengthy process.

4. Gene expression of oil palm roots in response to Ganoderma infection Disease resistant oil palms are crucial for sustainable production of palm oil. DNA markers that are linked to disease resistance in oil palm are required for screening and marker-assisted breeding of disease resistant oil palms but have not been reported so far. An in depth understanding of oil palm defence mechanisms in response to colonization of Ganoderma spp. at various stages of infection, are necessary at the transcript level to identify polygenic genes that contribute to a reasonable and durable disease resistance to Ganoderma spp. Knowledge on the molecular interactions between oil palm–Ganoderma may also offer alternatives to management of BSR disease. The oil palm molecular defence mechanisms in response to invasion of Ganoderma spp. can be elucidated by analysing the expression of individual genes that are homologous to genes reported as involved in defense pathway in other plants, or by analysing the global gene expression of the total mRNA populations. The former approach involves fewer genes, is less costly, less time consuming and facilitates the profiling of targeted transcripts only; while the latter approach involves a collection of transcripts that represent the total mRNA, is in large scale, costly, needs development of tools/facilities and is able to provide global information on gene or protein networks in the plant materials. Both approaches have been used by researchers to study the gene expression of oil palm in response to controlled inoculation with Ganoderma spp. Prior to the genome sequencing of oil palm and its deposition in the public database, global analysis of oil palm gene expression relied on information generated by expressed sequence tags (ESTs) which are short cDNA sequences generated and sequenced from the mRNA of oil palm. In the last decade, a total number of 40,809 ESTs from oil palm have been generated from various oil palm tissues and deposited at the EST database by various groups of researchers (www.ncbi.nlm.nih.gov/genbank/dbest/, Jouannic et al., 2005; Ho et al., 2007; Low et al., 2008). The generation of ESTs has facilitated the development and fabrication of a cDNA microarray consisting of more than 3700 cDNA probes for gene expression study of oil palm (Lim et al., 2010; Tee et al., 2013). In the preparation of cDNA microarray probes, cDNA sequences

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amplified by PCR are fixed onto a microscopic slide with a chemically treated surface. Fluorescent-labelled mRNA samples extracted from un-inoculated oil palm seedlings and oil palm seedlings inoculated with Ganoderma, respectively, can then hybridise with the cDNA microarray. By measuring and analysing the fluorescence emitted by mRNAs that are complementary to individual cDNA probes, the gene expression level of individual sequences can be profiled and compared between un-inoculated oil palm seedlings and oil palm seedlings inoculated with Ganoderma. Using this approach, Tee et al. (2013) reported a total of 61 transcripts being differentially expressed (up-regulated more than twofold or down-regulated less than twofold) in oil palm roots that have been artificially inoculated with G. boninense for 3 and 6 weeks, respectively, compared to those from un-inoculated roots. Upon Ganoderma inoculation, oil palm genes could either be upregulated in infected oil palms to strengthen the host defense against fungal invasion including genes encoding pathogenesisrelated protein 1, heat-shock protein-70 cofactor, isoflavone reductase, early-methionine-labelled polypeptides and early nodulin-20); or be down-regulated due to the suppression of the host defense system by the pathogen such as genes encoding vicilin-like antimicrobial peptide, pecanex-like protein and extension-1 (Tee et al., 2013). Some of these genes that were differentially expressed in inoculated oil palms are summarized in Fig. 1. Similar to other plants, oil palm can activate inducible defence response to pathogenic invasion; however, the effectiveness of these responses against Ganoderma is unknown. Although global gene expression analysis has been initiated to analyse the defence response of oil palm to Ganoderma, the study by Tee et al. (2013) suffered a major limitation whereby only a small number of gene sequences (approximately 3000 genes) were included in the cDNA microarray, and therefore could not provide a complete picture of the molecular defence response of oil palm against Ganoderma infection. We still do not have answers to many questions, for example, what are the oil palm defence genes and mechanisms besides those already known and reviewed here? Although the general defence pathways are well characterized in higher plants, the applicability of those models developed for disease resistance in model plants has to be evaluated in oil palm. In addition, there are specific questions that need to be answered; for example, can oil palm switch its defense responses rapidly to Ganoderma spp. at biotrophic and necrotrophic phases, through the interplay of hormones? What are the associations of HR-associated cell death and the kinetics of oxidative burst to the susceptibility of oil palm to Ganoderma spp.? Can oil palm produce antimicrobial compounds at the site of infection to suppress the spread of Ganoderma spp. (Fig. 1)? The availability of the oil palm genome information, cheaper and faster sequencing service will contribute to the global gene expression profiling of oil palm genes in response to the pathogen soon. In addition, oil palm ESTs have also provided information for molecular cloning and characterization of targeted genes. A total of 22 oil palm genes including those encoding PR proteins i.e., glucanases, chitinases, proteinase-inhibitors, defensin and isoflavone reductase have been identified from the oil palm ESTs for sequence analysis and gene expression profiling (Naher et al., 2011; Yeoh et al., 2012, 2013; Tan et al., 2013; Tee et al., 2013). In this approach, the gene expression level of individual sequences can be profiled and compared between un-inoculated oil palm seedlings and oil palm seedlings inoculated with Ganoderma by quantitative reverse-transcription (qRT)-PCR (Naher et al., 2011; Yeoh et al., 2012, 2013; Tan et al., 2013; Tee et al., 2013) or semi-quantitative RT-PCR (Alizadeh et al., 2011). Induction of pathogenesis-related proteins (PRs) has been found in many plant species belonging to various families (van Loon, 1999; van Loon and van Strien, 1999). Currently, there are 17


6


pathogen

ROS SOD

Strengthening of cell wall effector

CWDEs from pathogen PAMPs

pathogen

DAMPs Cell wall Cell membrane

R protein? Phytotoxic ? compounds? ? ?

Rboh

ROS

?

Ethylene

?

SA

Phe PAL CA

? Disease- and HRassociated cell death

PRR

JA

IFR Phenolic accumulation

Phytoalexin accumulation

Regulation of gene expression

PR proteins: Proteinase inhibitors, defensins, pathogenesis related-1 glucanases chitinases

Stress-related proteins Transcription factor UNE10, HSP-70 cofactor, metallothionien-like, Em protein H2, EMZ08, Extensin , extracellular ribonuclease, SPX-domain containing protein, vicilin-like antimicrobial peptidase

Other proteins Auxin-responsive protein IAA7, wall-associated receptor kinase, sedoheptulose-1,7-biphosphate, early nodulin, IFR YABBY 2, pecanex-like protein, alpha ketoglutarate-dependent dioxygenase, PAL

Fig. 1. Proposed oil palm–Ganoderma molecular interactions and summary of oil palm genes that respond to the infection by Ganoderma boninense. At an early stage of interaction, fungal pathogen may release pathogen associated molecular patterns (PAMPs) which are recognized by the pattern recognition receptors (PRRs) residing in the cell membrane. In the necrotrophic phase, the fungal pathogen secretes cell wall degrading enzymes (CWDEs) that degrade cell wall of plant host (including lignin) producing fragments of structural polysaccharides known as damage associated molecular patterns (DAMPs). The recognition of P/DAMPs by PRRs in the host plant converges into a few signalling pathways that regulate transcription of defense genes in oil palm including those encoding pathogenesis-related (PR) proteins, stress proteins and other proteins in the cell. It is unknown whether an effector is released by Ganoderma spp. during its biotrophic phase, which could possibly interact with R protein and elicit HR-associated cell death via the production of reactive oxygen species (ROS). ROS could also induce disease-associated cell death during necrotrophic phase. The ability of Ganoderma spp. to produce phytotoxin is unknown. Blue arrows in broken line indicate multiple steps in a pathway. Brown arrows in broken line represent the possible signalling pathways while the black arrows in broken line represent the roles of protein encoded by some of the up-regulated or down-regulated genes. The blue dotted line arrows show the possible defence responses in oil palm, whereas the black dotted lines arrows indicate possible interactions (synergy between SA and ethylene, and antagonism between SA and JA) between hormones. The question marks highlight the gaps that need to be filled in oil palm molecular defences responses to Ganoderma spp. The words in red and green indicate plant genes that were up-regulated and down-regulated in Ganoderma-inoculated oil palm in comparison to the un-inoculated oil palm root tissues, respectively. CA, cinnamate; HR, hypersensitive reaction; IFR, isoflavone reductase; JA, jasmonate; PAL, phenylalanine ammonia-lyase; Phe, phenylalanine; ROS, reactive oxygen species; SA, salicylate; SOD, superoxide dismutase. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

families of PR proteins: PR-1 (antifungal), PR-2 (b-1,3-glucanase), PR-3 (chitinase type I, II, IV, V, VI, VII), PR-4 (chitinase type I, II), PR-5 (thaumatin-like), PR-6 (proteinase-inhibitor), PR-7 (endoproteinase), PR-8 (chitinase type III), PR-9 (peroxidase), PR-10 (ribonuclease-like), PR-11 (chitinase type I), PR-12 (defensin), PR-13 (thionin), PR-14 (lipid-transfer protein), PR-15 (oxalate oxidase), PR-16 (‘oxalate oxidase-like’), PR-17 (unknown), with most of their activities known (van Loon and van Strien, 1999). The structural and functional characteristics of each class were well reviewed (Selitrennikoff, 2001) and will not be discussed here. In this review, the expression of genes encoding PR proteins and stress related proteins in Ganoderma-inoculated oil palm tissues, and their possible roles are summarized (Table 1 and Fig. 1). Only the changes in gene expression level in Ganoderma-infected oil palm tissues in comparison to un-inoculated oil palm tissues that are equal or more than 2-fold will be considered. The genes were up-regulated possibly as part of plant defence against Ganoderma spp. whereas the down-regulated genes in oil palm roots were possibly suppressed by the pathogen. Discussion on individual gene expression profiles has been detailed by individual references and thus will not be repeated here. Many of these genes (Table 1) display transient up- or down-regulation at different stages of infection thereby complicating the analysis of gene expression profiles. Nevertheless, transcript profiling of some of these genes belonging to a

big family, especially genes encoding chitinases and glucanases from oil palm, has enabled identification of specific isoforms related to pathogenesis (Naher et al., 2011; Yeoh et al., 2012, 2013). This is especially important for isozymes that may have many functions other than that in plant defense. Expression analyses of genes encoding enzymes related to phenylpropanoid and isoflavonoid phytoalexin pathways (Table 1) are important to understand the molecular defense of oil palm against Ganoderma spp. since many flavonoids and isoflavonoid are important antimicrobial compounds (Kramr et al., 1984; Cruickshank, 1962). The expression of an oil palm gene encoding isoflavone reductase (EgIFR) was higher in roots inoculated with G. boninense compared to that of the un-inoculated roots (Tee et al., 2013) suggesting that the biosynthesis isoflavonoid phytoalexin could be induced by this pathogen in oil palm. Furthermore, the transcript profiles of EgPAL and EgC4H which encode L-phenylalanine lyase and cinnamate 4-hydroxylase, respectively, may provide more information on the biosynthesis of lignin precursors i.e. cinnamate and p-coumarate. The transcript abundance of EgPAL and EgC4H was down-regulated in oil palm roots at an early stage of G. boninense inoculation, supporting the finding of Rees et al. (2009) that oil palm may not rely on wall-associated responses against invasion of Ganoderma spp. Instead, oil palm may be more reliant on the production of antimicrobial compounds against the fungal


Transcripts

Putative functions

Abbreviation

Gene expression profiles in inoculated oil palm seedlings compared with uninoculated oil palm seedlings

References

Glucanases b-D-glucan exohydrolase Glucan endo-1,3-Dglucosidase

PR-2; degrade fungal cell wall component by hydrolyzing b-1,3glucosidic linkages and promote the release of cell-wall derived fungal elicitors

EgGlc1-1 EgGlc5-1

Up-regulated in the leaves of inoculated oil palm seedlings at 3 and 12 wpi Down-regulated in the roots of inoculated oil palm seedlings from 3, 6 and12 wpi, and leaves of inoculated oil palm seedlings at 6 wpi Down-regulated in the roots of inoculated oil palm seedlings from 3, 6 and12 wpi, suppressed in the leaves of inoculated oil palm seedlings at 6 and12 wpi; up-regulated in the leaves of inoculated oil palm seedlings at 3 wpi

Yeoh et al. (2012)

Up-regulated in the roots of inoculated oil palm seedlings at 5 wpi Up-regulated in the leaves of inoculated oil palm seedlings at 3 wpi; suppressed in the roots and leaves of inoculated oil palm seedlings at 6 and 12 wpi Up-regulated in the roots of inoculated oil palm seedlings at 2 and 5 wpi; Up-regulated in the roots of inoculated oil palm seedlings at 5 wpi Up-regulated in the roots of inoculated oil palm seedlings at 12 wpi, and leaves of inoculated oil palm seedlings at 6 and 12 wpi; down-regulated in the roots and leaves of inoculated oil palm seedlings at 3 and 6 wpi Up-regulated in the leaves of inoculated oil palm seedlings at 3 wpi, downregulated in the roots at 6 wpi and leaves of inoculated oil palm seedlings at 3 wpi

Naher et al. (2011) Yeoh et al. (2013)

Up-regulated in the roots of inoculated oil palm seedlings at 12 wpi and leaves of inoculated oil palm seedlings at 3 wpi Up-regulated in the roots of inoculated oil palm seedlings at 3 and 6 wpi and leaves of inoculated oil palm seedlings at 6 wpi Up-regulated in the roots of inoculated oil palm seedlings at 12 wpi; downregulated in the roots of inoculated oil palm seedlings at 6 wpi

Tan et al. (2013)

Down-regulated in the roots of inoculated oil palm seedlings at 12 wpi and leaves at 6 and 12 wpi; up-regulated in the leaves of inoculated oil palm seedlings at 3 wpi Up-regulated in the roots of inoculated oil palm seedlings at 12 wpi but down-regulated in roots at 3 wpi; up-regulated in the leaves of inoculated oil palm seedlings at 3 wpi

Tan et al. (2013)

Up-regulated in the roots of inoculated oil palm seedlings at 21 dpi; induced in the leaves of inoculated oil palm seedlings at 3 dpi Induced in the leaves of inoculated oil palm seedlings at 7 dpi Up-regulated in the roots of inoculated oil palm seedlings at 12 wpi; downregulated in the roots of inoculated oil palm seedlings at 3 and 6 wpi

Alizadeh et al. (2011)

Up-regulated in the roots of inoculated oil palm seedlings at 6 wpi, downregulated in the leaves of inoculated oil palm seedlings at 3,6 and12 wpi Up-and down-regulation less than 2-fold in all tissues examined Up-regulated in the roots of inoculated oil palm seedlings at 3, 6 and 12 wpi and leaves at 3 wpi

Tan et al. (2013)

Up-regulated in the roots of inoculated oil palm seedlings at 6 wpi Up-and down-regulation less than 2-fold in all tissues examined Down-regulated in the roots of inoculated oil palm seedlings at 3 wpi

Tee et al. (2013) Tan et al. (2013) Tee et al. (2013)

EgGlc5-2

Chitinases/chitinase-like Class I chitinase

PR-3, PR-4, PR-8 and PR-11; cleave the b-1,4-glycosidic linkages between N-acetylglucosamine residues in fungal chitin to chitin oligosaccharides

EgCHI1 EgChit1-1

Class II chitinase Class III chitinase

EgCHI2 EgCHI3 EgChit3-1

Class V chitinase

EgChit5-1

Proteinase-inhibitor Bowman–Birk serine protease inhibitor

PR-6; serine proteinase inhibitors that specifically regulate proteinases belonging to serine classes

EgBBI1 EgBBI2

Defensin

Pathogenesis-related 1 protein

PR-12; cysteine-rich proteins with many different modes of actions, including anti-microbial, anti-fungal, insecticidal, protease inhibiting, and a-amylase inhibiting activities (Stotz et al., 2009) PR-1; antifungal with unknown modes of action, cellular and molecular targets

EgDFS

EgPRP

Ribosome-inactivating protein

Ribotoxins, type 2 ribosome-inactivating protein

EgT2RIP

Metallothioneins

Intracellular cysteine-rich metal-binding proteins. with reactive oxygen species scavenging activities

MT3-A MT3-B EgMT

Late embryogenesis associated proteins Related to development and abiotic stresses Early methionine-labeled polypeptide (group 1 LEA) Dehydrin (group 2 LEA)

EgEMLP2 EgDHN

Enzymes related to phenylpropanoid and isoflavonoid phytoalexin pathways Isoflavone reductase Biosynthesis of isoflavonoid phytoalexin

EgIFR

L-Phenylalanine

lyase

Catalyzes the biosynthesis of cinnamate

EgEMLP1

EgPAL

Naher et al. (2011) Naher et al. (2011) Yeoh et al. (2013)

Yeoh et al. (2013)

Tan et al. (2013)

Tan et al. (2013)


Alizadeh et al. (2011) Tan et al. (2013)

Tan et al. (2013)

(continued on next page) 7


Table 1 A summary of gene expression profiles of individual defense related genes in inoculated oil palm seedlings compared with un-inoculated oil palm seedlings.



invasion, which happens at a later stage as evident by the gene expression profile of EgIFR (Table 1). So far, molecular identification and characterization of oil palm defense genes have been carried out on oil palm seedlings derived from tenera seeds with different degrees of susceptibility to Ganoderma spp., and thus may limit the identification of major genes that contribute to resistance/tolerance. Expansion of these studies to clonal oil palms that have been proven to be tolerant/resistant to Ganoderma spp. may provide more consistent and remarkable differences in gene expression profiles that make the identification of resistance genes easier. However, oil palm with an ‘‘all-ornothing type’’ of resistance has not been reported. In addition, the reproducibility of artificial inoculations of oil palm materials with Ganoderma spp. is important for gene expression analysis. Reliable methods which can detect infection stages of oil palm roots without uplifting the plants from the soil are required. In most of the previous studies, destructive sampling has been practised and the oil palm seedlings were harvested according to the duration of inoculation instead of the stage of infection. The duration of inoculation is not a good indicator for infection stage because the levels of infection can be easily affected by the age of the plants, size and virulence of inoculum, shading and temperature (Rees et al., 2007). Due to the non-synchronous nature of infection of Ganoderma spp., the tissues collected were not specific to a particular stage of infection alone, in fact they were either a mixture of healthy and necrotic tissues, or a mixture of tissues at various infection stages most of the time. The difficulties encountered in correlating temporal gene expression to infection stages have to be overcome.

Up-regulated in the roots of inoculated oil palm seedlings at 21 dpi; downregulated in the leaves of inoculated oil palm seedlings at 42 and 63 dpi Up-regulated in the leaves of inoculated oil palm seedlings at 21 dpi SAD1

4. Conclusions

wpi, weeks post inoculation; dpi, days post inoculation.

SAD1

Tee et al. (2013) EgCHI Catalyzes chalcone to flavone Chalcone flavone isomerase

D9 stearoyl-acyl carrier protein desaturase Catalyzes conversion of saturated stearic acid to mono-saturated oleic Oil palm D9 stearoyl-acyl acid; regulates the defence response of plants to pathogens by carrier protein inducing salicyclic acid and jasmonic acid-mediated defence response desaturase

Tee et al. (2013)

Down-regulated in the roots of inoculated oil palm seedlings at 3 wpi; upregulated in the roots of inoculated oil palm seedlings at 12 wpi Down-regulated in the roots of inoculated oil palm seedlings at 3 wpi Catalyzes cinnamate to p-coumarate Cinnamate 4-hydrolase

EgC4H

Putative functions Transcripts

Table 1 (continued)

Abbreviation

References


Gene expression profiles in inoculated oil palm seedlings compared with uninoculated oil palm seedlings

8

In recent years, comparative analyses of global gene expression and transcript profiles of oil palm PR proteins in inoculated and uninoculated oil palm seedlings have started to reveal the molecular defence mechanisms of oil palm against Ganoderma spp. Findings on the gene regulation of oil palm defence pathways although fragmentary have brought us a step closer toward the understanding of polygenic resistance of oil palm against Ganoderma spp. Polygenic resistance comprising many genes of minor effect may provide reasonable levels of resistance in oil palm. Identification and characterization of genes that contribute to this polygenic resistance could be useful for screening for disease resistant oil palm and marker-assisted breeding programme. Knowledge on associations of HR-induced cell death and ROS kinetics, to the susceptibility of oil palm to Ganoderma spp.; the interactions of phytohormones (salicylate, jasmonate and ethylene) at early and late stages of BSR; and cell wall strengthening through increased production of Gtype lignin; are required to understand the molecular interactions between oil palm and Ganoderma spp. The advance of new generation of sequencing technologies and availability of oil palm genome information will hasten the pace of gene expression profiling in oil palm and Ganoderma spp. in the near future. The lignolytic activities of plant CWDEs from Ganoderma spp. on different types of lignin are obscure, and await further investigations. The breeding of disease resistant oil palms should be considered despite the lengthy process required. The issues of acceptability of consumers to genetically manipulated oil palm have to be considered prior to genetic modification of oil palm with higher resistance to Ganoderma spp. Acknowledgements This project was funded by RUGS Initiative 6 UPM (05-02-111408RU) and Putra Grant (GP-IPB/2013/9413601). Tan Y.-C. was supported by Malaysia Ministry of Science, Technology and



Innovation under National Science Fellowship (NSF). We thank the reviewers for their constructive comments.

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C.-L. Ho is currently an associate professor at the Faculty of Biotechnology and Biomolecular Sciences in University Putra Malaysia. She obtained her PhD from the Faculty of Pharmaceutical Sciences, Chiba University, Japan. Her current research is focused on oil palm defense responses to Ganoderma sp. and transcriptomes of an agarophyte, Gracilaria changii. She has published more than 60 peer-reviewed research and review papers.

Y.-C. Tan graduated in 2008 from Universiti Malaysia Sabah with a BSc. degree in Biotechnology. After graduation, he worked in Nimura Genetic Solutions (M) Sdn. Bhd., on bioprospecting and drugs discovery from soil bacteria (actinomycetes) and fungi. Then, he pursued his MSc degree in the field of Molecular Biology at Universiti Putra Malaysia and graduated in 2013. He studied some of the host defence-related genes in oil palm when exposed to pathogenic fungus Ganoderma sp. He is currently working as a genome informaticist in Codon Genomics Sdn. Bhd. analyzing big data from next generation sequencing.


The Phenylpropanoid Pathway and Lignin in Defense against Ganoderma boninense Colonized Root Tissues in Oil Palm (Elaeis guineensis Jacq.).

Mating Compatibility and Restriction Analysis of Ganoderma Isolates from Oil Palm and Other Palm Hosts.

Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

Identification of proteins of altered abundance in oil palm infected with Ganoderma boninense.

Commercially available alternatives to palm oil.

Scytalidium parasiticum sp. nov., a New Species Parasitizing on Ganoderma boninense Isolated from Oil Palm in Peninsular Malaysia.

Introduction: nutritional aspects of palm oil.

Analyses of hypomethylated oil palm gene space.

Key glycolytic branch influences mesocarp oil content in oil palm.

India has a problem with palm oil.

Palm oil and the heart: A review.

Comparative alteration in atherogenic indices and hypocholesteremic effect of palm oil and palm oil mill effluent in normal albino rats.

Trace elements and radionuclides in palm oil, soil, water, and leaves from oil palm plantations: A review.

Oil body-mediated defense against fungi: From tissues to ecology.

Genetic architecture of palm oil fatty acid composition in cultivated oil palm (Elaeis guineensis Jacq.) compared to its wild relative E. oleifera (H.B.K) Cortés.

Inhibition of palm oil oxidation by zeolite nanocrystals.

Nutritional evaluation of crude palm oil in rats.

Life Cycle Assessment for the Production of Oil Palm Seeds.

Nonhypercholesterolemic effects of a palm-oil diet in Malaysian volunteers.

Microwave-enhanced CO2 gasification of oil palm shell char.

Cardioprotective effect of virgin coconut oil in heated palm oil diet-induced hypertensive rats.

Evaluation of Palm Oil as a Suitable Vegetable Oil for Vitamin A Fortification Programs.

Synthesis and characterization of polyacids from palm acid oil and sunflower oil via addition reaction.

Utilization of Palm Oil Clinker as Cement Replacement Material.