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Risk factors for endemic giardiasis: highlighting the possible association of contaminated water and food

Mohammed Mahdy AK, Lim YA, Surin J, Wan KL, Al-Mekhlafi MS

Trans R Soc Trop Med Hyg, 2008 May;102(5):465-70.
PMID: 18377940 DOI: 10.1016/j.trstmh.2008.02.004

This study was conducted to reassess the risk factors for giardiasis in communities of the Orang Asli (indigenous people) in Pahang, Malaysia. Stool samples were collected from 321 individuals (2-76 years old; 160 males, 161 females). Data were collected via laboratory analysis of faecal samples and a pre-tested standard questionnaire. River water samples were tested for Giardia cysts and Cryptosporidium oocysts. The overall prevalence of G. intestinalis infection was 23.7%. Children < or =12 years old had the highest infection rate and have been identified as a high risk group (odds ratio (OR)=6.2, 95% CI 1.5-27.0, P<0.005). The risk of getting giardiasis also appeared to be significantly associated with drinking piped water (OR=5.1, 95% CI 0.06-0.7, P<0.005) and eating raw vegetables (OR=2.4, 95% CI 0.2-0.6, P<0.005). In conclusion, sociodemographic factors have always been associated with the high prevalence of Giardia infections in Malaysia. However, the present study also highlights the need to look into the possibility of other risks such as water and food transmission routes. In future, it is necessary that these two aspects be considered in control strategies.
Identification and analysis of a prepro-chicken gonadotropin releasing hormone II (preprocGnRH-II) precursor in the Asian seabass, Lates calcarifer, based on an EST-based assessment of its brain transcriptome

Tan SL, Mohd-Adnan A, Mohd-Yusof NY, Forstner MR, Wan KL

Gene, 2008 Mar 31;411(1-2):77-86.
PMID: 18280674 DOI: 10.1016/j.gene.2008.01.008

Using a novel library of 5637 expressed sequence tags (ESTs) from the brain tissue of the Asian seabass (Lates calcarifer), we first characterized the brain transcriptome for this economically important species. The ESTs generated from the brain of L. calcarifer yielded 2410 unique transcripts (UTs) which comprise of 982 consensi and 1428 singletons. Based on database similarity, 1005 UTs (41.7%) can be assigned putative functions and were grouped into 12 functional categories related to the brain function. Amongst others, we have identified genes that are putatively involved in energy metabolism, ion pumps and channels, synapse related genes, neurotransmitter and its receptors, stress induced genes and hormone related genes. Subsequently we selected a putative preprocGnRH-II precursor for further characterization. The complete cDNA sequence of the gene obtained was found to code for an 85-amino acid polypeptide that significantly matched preprocGnRH-II precursor sequences from other vertebrates, and possesses structural characteristics that are similar to that of other species, consisting of a signal peptide (23 residues), a GnRH decapeptide (10 residues), an amidation/proteolytic-processing signal (glycine-lysine-argine) and a GnRH associated peptide (GAP) (49 residues). Phylogenetic analysis showed that this putative L. calcarifer preprocGnRH-II sequence is a member of the subcohort Euteleostei and divergent from the sequences of the subcohort Otocephalan. These findings provide compelling evidence that the putative L. calcarifer preprocGnRH-II precursor obtained in this study is orthologous to that of other vertebrates. The functional prediction of this preprocGnRH-II precursor sequence through in silico analyses emphasizes the effectiveness of the EST approach in gene identification in L. calcarifer.
In silico identification and characterization of a putative phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gene in Eimeria tenella

Ling KH, Loo SS, Rosli R, Shamsudin MN, Mohamed R, Wan KL

In Silico Biol. (Gedrukt), 2007;7(1):115-21.
PMID: 17688436

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) play diverse roles in the cellular biology of many organisms, including signal transduction, secretion and vesicular trafficking, and regulation of cytoskeleton assembly. Discovery of the PIP5K gene in Eimeria tenella may shed light on its role in the biology of this avian protozoan, and afford further understanding of the cell-host interaction, particularly during the invasion process. In this study, we report the identification of the PIP5K coding region in the genome sequence of Eimeria tenella using in silico gene prediction approaches. Prediction of the PIP5K coding sequence was confirmed by mapping the full-length cDNA sequence, generated via the Rapid Amplification of cDNA Ends (RACE) method, to the genomic sequence. The putative PIP5K gene of Eimeria tenella is located on the complementary strand of the E1080B12.b1 contig, and comprises 12 exons. Further analysis showed that the coding region spans from exon 1 to exon 7, with all exons obeying the adopted 'gt...ag' splicing rule of intronic sequences. Consensus of the hexameric 5' donor-splice site was deduced as GTRDBB... and the consensus for the 3' acceptor-splice sites as ...BHDYAG. The gene encodes a 252-amino acid residue protein. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a PIP5K.
Development of a modified method for sequencing high G:C regions in chicken anemia virus genome

Chowdhury SM, Omar AR, Aini I, Hair-Bejo M, Jamaluddin AA, Wan KL, et al.

J. Biochem. Mol. Biol. Biophys., 2002 Jun;6(3):229-32.
PMID: 12186760

Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
Comparative EST analyses provide insights into gene expression in two asexual developmental stages of Eimeria tenella

Ng ST, Sanusi Jangi M, Shirley MW, Tomley FM, Wan KL

Exp Parasitol, 2002 11 13;101(2-3):168-73.
PMID: 12427472

The protozoan parasite Eimeria tenella has a complex life cycle that includes two major asexual developmental stages, the merozoite and the sporozoite. The expressed sequence tag (EST) approach has been previously used to study gene expression of merozoites. We report here the generation and analysis of 556 ESTs from sporozoites. Comparative analyses of the two datasets reveal a number of transcripts that are preferentially expressed in a specific stage, including previously uncharacterised sequences. The data presented indicate the invaluable potential of the comparative EST analysis for providing information on gene expression patterns in the different developmental stages of E. tenella.
A survey of genes in Eimeria tenella merozoites by EST sequencing

Wan KL, Chong SP, Ng ST, Shirley MW, Tomley FM, Jangi MS

Int J Parasitol, 1999 Dec;29(12):1885-92.
PMID: 10961844

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.
Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex

Chow KS, Wan KL, Isa MN, Bahari A, Tan SH, Harikrishna K, et al.

J Exp Bot, 2007;58(10):2429-40.
PMID: 17545224

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.
Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia

Siti-Futri FF, Rosilawati R, Wan KL, Cheong YL, Nazni WA, Lee HL

Trop Biomed, 2020 Mar 01;37(1):201-209.
PMID: 33612731

The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.
Fulltext Three-year follow-up of a Traumatic Hemipelvectomy Survivor: A Case Report

Wan KL, Azlan MS, Syed-Azmi AS, Lattish R, Faisham WI

Malays Orthop J, 2021 Nov;15(3):143-146.
PMID: 34966511 DOI: 10.5704/MOJ.2111.024

The management of a patient with traumatic hemipelvectomy is complex. We report the acute management and rehabilitation of a 21-year-old patient as well as her prosthesis modification. She was able to return to society as a K3 level ambulator.
Eimeria maxima phosphatidylinositol 4-phosphate 5-kinase: locus sequencing, characterization, and cross-phylum comparison

Goh MY, Pan MZ, Blake DP, Wan KL, Song BK

Parasitol Res, 2011 Mar;108(3):611-20.
PMID: 20938684 DOI: 10.1007/s00436-010-2104-7

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) may play an important role in host-cell invasion by the Eimeria species, protozoan parasites which can cause severe intestinal disease in livestock. Here, we report the structural organization of the PIP5K gene in Eimeria maxima (Weybridge strain). Two E. maxima BAC clones carrying the E. maxima PIP5K (EmPIP5K) coding sequences were selected for shotgun sequencing, yielding a 9.1-kb genomic segment. The EmPIP5K coding region was initially identified using in silico gene-prediction approaches and subsequently confirmed by mapping rapid amplification of cDNA ends and RT-PCR-generated cDNA sequence to its genomic segment. The putative EmPIP5K gene was located at position 710-8036 nt on the complimentary strand and comprised of 23 exons. Alignment of the 1147 amino acid sequence with previously annotated PIP5K proteins from other Apicomplexa species detected three conserved motifs encompassing the kinase core domain, which has been shown by previous protein deletion studies to be necessary for PIP5K protein function. Phylogenetic analysis provided further evidence that the putative EmPIP5K protein is orthologous to that of other Apicomplexa. Subsequent comparative gene structure characterization revealed events of intron loss/gain throughout the evolution of the apicomplexan PIP5K gene. Further scrutiny of the genomic structure revealed a possible trend towards "intron gain" between two of the motif regions. Our findings offer preliminary insights into the structural variations that have occurred during the evolution of the PIP5K locus and may aid in understanding the functional role of this gene in the cellular biology of apicomplexan parasites.
Dissecting the Biology of Rafflesia Species: Current Progress and Future Directions Made Possible with High-Throughput Sequencing Data

Mursyidah AK, Hafizzudin-Fedeli M, Nor Muhammad NA, Latiff A, Firdaus-Raih M, Wan KL

Plant Cell Physiol, 2023 Apr 17;64(4):368-377.
PMID: 36611267 DOI: 10.1093/pcp/pcad004

The angiosperm Rafflesia exhibits a unique biology, including a growth strategy that involves endophytic parasitism of a specific host, with only the gigantic flower externally visible. The Rafflesia possesses many unique evolutionary, developmental and morphological features that are rooted in yet-to-be-explained physiological processes. Although studies on the molecular biology of Rafflesia are limited by sampling difficulties due to its rarity in the wild and the short life span of its flower, current advances in high-throughput sequencing technology have allowed for the genome- and transcriptome-level dissection of the molecular mechanisms behind the unique characteristics of this parasitic plant. In this review, we summarize major findings on the cryptic biology of Rafflesia and provide insights into future research directions. The wealth of data obtained can improve our understanding of Rafflesia species and contribute toward the conservation strategy of this endangered plant.
Fulltext Characterisation of full-length cDNA sequences provides insights into the Eimeria tenella transcriptome

Amiruddin N, Lee XW, Blake DP, Suzuki Y, Tay YL, Lim LS, et al.

BMC Genomics, 2012 Jan 13;13:21.
PMID: 22244352 DOI: 10.1186/1471-2164-13-21

BACKGROUND: Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis.
RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.
CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.
Fulltext RNA-seq data from different developmental stages of Rafflesia cantleyi floral buds

Amini S, Alias H, Aizat-Juhari MA, Mat-Isa MN, Adam JH, Goh HH, et al.

Genom Data, 2017 Dec;14:5-6.
PMID: 28761813 DOI: 10.1016/j.gdata.2017.07.008

Rafflesia cantleyi, known as one of the world's largest flowers, is a specialised holoparasite due to dramatic morphological modifications. It possesses highly reduced vegetative structure and only appears as a flower for sexual reproduction. Moreover, it has an unusual life cycle in that its floral bud development takes up to nine months. In order to fully understand the highly modified floral organ structure and long life cycle of R. cantleyi, we used Illumina sequencing technology (HiSeq) for sequence generation followed by de novo assembly of sequence reads. We obtained the RNA-seq data from three different stages of floral bud, representing the early, mid and advanced developmental stages. These data are available via BioProject accession number PRJNA378435. More than 10.3 Gb raw sequence data were generated, corresponding to 102,203,042 raw reads. Following removal of low-quality reads and trimming of adapter sequences, a total of 91,638,836 reads were obtained. De novo assembly of these sequences using Trinity resulted in 89,690 unique transcripts with an N50 of 1653 bp. The obtained transcriptomic data will be useful for further study to understand the molecular interactions that result in R. cantleyi floral development.
Fulltext Comparative analysis of nucleus-encoded plastid-targeting proteins in Rafflesia cantleyi against photosynthetic and non-photosynthetic representatives reveals orthologous systems with potentially divergent functions

Ng SM, Lee XW, Mat-Isa MN, Aizat-Juhari MA, Adam JH, Mohamed R, et al.

Sci Rep, 2018 Nov 22;8(1):17258.
PMID: 30467394 DOI: 10.1038/s41598-018-35173-1

Parasitic plants are known to discard photosynthesis thus leading to the deletion or loss of the plastid genes. Despite plastid genome reduction in non-photosynthetic plants, some nucleus-encoded proteins are transported back to the plastid to carry out specific functions. In this work, we study such proteins in Rafflesia cantleyi, a member of the holoparasitic genus well-known for producing the largest single flower in the world. Our analyses of three transcriptome datasets, two holoparasites (R. cantleyi and Phelipanche aegyptiaca) and one photosynthetic plant (Arabidopsis thaliana), suggest that holoparasites, such as R. cantleyi, retain some common plastid associated processes such as biosynthesis of amino acids and lipids, but are missing photosynthesis components that can be extensions of these pathways. The reconstruction of two selected biosynthetic pathways involving plastids correlates the trend of plastid retention to pathway complexity - transcriptome evidence for R. cantleyi suggests alternate mechanisms in regulating the plastidial heme and terpenoid backbone biosynthesis pathways. The evolution to holoparasitism from autotrophy trends towards devolving the plastid genes to the nuclear genome despite the functional sites remaining in the plastid, or maintaining non-photosynthetic processes in the plastid, before the eventual loss of the plastid and any site dependent functions.
Fulltext Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex

Chow KS, Mat-Isa MN, Bahari A, Ghazali AK, Alias H, Mohd-Zainuddin Z, et al.

J Exp Bot, 2012 Mar;63(5):1863-71.
PMID: 22162870 DOI: 10.1093/jxb/err363

The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
Fulltext Perigone Lobe Transcriptome Analysis Provides Insights into Rafflesia cantleyi Flower Development

Lee XW, Mat-Isa MN, Mohd-Elias NA, Aizat-Juhari MA, Goh HH, Dear PH, et al.

PLoS One, 2016;11(12):e0167958.
PMID: 27977777 DOI: 10.1371/journal.pone.0167958

Rafflesia is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the Rafflesia cantleyi flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled de novo into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of R. cantleyi. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of R. cantleyi. Genes potentially involved in the growth and development of the R. cantleyi flower were identified and provide insights into biological processes that occur during flower development.
Fulltext Transcriptome landscape of Rafflesia cantleyi floral buds reveals insights into the roles of transcription factors and phytohormones in flower development

Amini S, Rosli K, Abu-Bakar MF, Alias H, Mat-Isa MN, Juhari MA, et al.

PLoS One, 2019;14(12):e0226338.
PMID: 31851702 DOI: 10.1371/journal.pone.0226338

Rafflesia possesses unique biological features and known primarily for producing the world's largest and existing as a single flower. However, to date, little is known about key regulators participating in Rafflesia flower development. In order to further understand the molecular mechanism that regulates Rafflesia cantleyi flower development, RNA-seq data from three developmental stages of floral bud, representing the floral organ primordia initiation, floral organ differentiation, and floral bud outgrowth, were analysed. A total of 89,890 transcripts were assembled of which up to 35% could be annotated based on homology search. Advanced transcriptome analysis using K-mean clustering on the differentially expressed genes (DEGs) was able to identify 12 expression clusters that reflect major trends and key transitional states, which correlate to specific developmental stages. Through this, comparative gene expression analysis of different floral bud stages identified various transcription factors related to flower development. The members of WRKY, NAC, bHLH, and MYB families are the most represented among the DEGs, suggesting their important function in flower development. Furthermore, pathway enrichment analysis also revealed DEGs that are involved in various phytohormone signal transduction events such as auxin and auxin transport, cytokinin and gibberellin biosynthesis. Results of this study imply that transcription factors and phytohormone signalling pathways play major role in Rafflesia floral bud development. This study provides an invaluable resource for molecular studies of the flower development process in Rafflesia and other plant species.
Fulltext The structure of a major surface antigen SAG19 from Eimeria tenella unifies the Eimeria SAG family

Ramly NZ, Dix SR, Ruzheinikov SN, Sedelnikova SE, Baker PJ, Chow YP, et al.

Commun Biol, 2021 03 19;4(1):376.
PMID: 33742128 DOI: 10.1038/s42003-021-01904-w

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αβα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.
Fulltext Transcriptome analysis of Rafflesia cantleyi flower stages reveals insights into the regulation of senescence

Mohd-Elias NA, Rosli K, Alias H, Juhari MA, Abu-Bakar MF, Md-Isa N, et al.

Sci Rep, 2021 Dec 08;11(1):23661.
PMID: 34880337 DOI: 10.1038/s41598-021-03028-x

Rafflesia is a unique plant species existing as a single flower and produces the largest flower in the world. While Rafflesia buds take up to 21 months to develop, its flowers bloom and wither within about a week. In this study, transcriptome analysis was carried out to shed light on the molecular mechanism of senescence in Rafflesia. A total of 53.3 million high quality reads were obtained from two Rafflesia cantleyi flower developmental stages and assembled to generate 64,152 unigenes. Analysis of this dataset showed that 5,166 unigenes were differentially expressed, in which 1,073 unigenes were identified as genes involved in flower senescence. Results revealed that as the flowers progress to senescence, more genes related to flower senescence were significantly over-represented compared to those related to plant growth and development. Senescence of the R. cantleyi flower activates senescence-associated genes in the transcription activity (members of the transcription factor families MYB, bHLH, NAC, and WRKY), nutrient remobilization (autophagy-related protein and transporter genes), and redox regulation (CATALASE). Most of the senescence-related genes were found to be differentially regulated, perhaps for the fine-tuning of various responses in the senescing R. cantleyi flower. Additionally, pathway analysis showed the activation of genes such as ETHYLENE RECEPTOR, ETHYLENE-INSENSITIVE 2, ETHYLENE-INSENSITIVE 3, and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR, indicating the possible involvement of the ethylene hormone response pathway in the regulation of R. cantleyi senescence. Our results provide a model of the molecular mechanism underlying R. cantleyi flower senescence, and contribute essential information towards further understanding the biology of the Rafflesiaceae family.
Fulltext Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: identifying genes associated with callogenesis and embryogenesis

Low ET, Alias H, Boon SH, Shariff EM, Tan CY, Ooi LC, et al.

BMC Plant Biol, 2008 May 29;8:62.
PMID: 18507865 DOI: 10.1186/1471-2229-8-62

BACKGROUND: Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes.
RESULTS: A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames.
CONCLUSION: This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.

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