RT-qPCR profiling of pathogenesis related genes in Musa acuminata cv. 'Berangan' seedlings challenged with Fusarium oxysporum F. SP. Cubense tropical race 4

The expression profile of pathogenesis related genes are signatures of an infection response in plant cells. Pathogenic infections can increase or reduce gene expression in a plant system in a relatively specific pattern. These expression patterns can be used as standards in pathogenicity studies and, where phenotypic expression is normally used to gauge a plants response to infection, it could additionally present a more rapid and early screening reference tool. Three genes: catalase (CAT), pathogen related protein (PR10), and phenylalanine ammonia (PAL) all implicitly implicated in the plant disease response pathway were targeted for analysis during the infection of Fusarium oxysporum f. sp. cubense tropical race 4 (FOCR4) in banana Musa acuminata cv. Berangan seedlings after a standard challenge under growth room conditions. Distinct patterns of gene expression were observed at three infection time points by real time expression analysis. There was a sequential 10-fold reduction in expression for the PR gene while, the PAL and CAT genes were both upregulated. These results present a set of reference genes that could be used for screening of a plant’s response to Fusarium before the onset of symptoms

Elucidation of Musa acuminata cv. Berangan root infection by FOC (Tropical Race 4) by RNA sequencing and analysis

Musa acuminata cv. Berangan (AAA) is a type of banana locally grown in Malaysia. These bananas as well as Musa acuminata cv. Cavendish (AAA) are also facing a major threat from a typical soil borne fungus identified as Fusarium oxysporum f. sp. cubense race 4 (FocR4). Its characteristics as a complex pathogen manifesting as subtypes or races are the main reasons its infections are difficult to control. Genome sequence availability of the double haploid Musa acuminata originating from Pahang has become very useful to analyse RNA-seq reads and to identify the transcriptome profile of the host response between different groups was accomplished using RNA-Seq technology based on the Illumina HiSeqTm 2000 platform. Three sets of libraries derived from infected and mock infected plants (experimental groups) between different time points (0, 48, 96 h) shows over forty million reads were generated, each corresponding to coverage of >4,000,000,000 to <8,000,000,000 bases. About 0.10-66% reads were mapped to Musa acuminata DH Pahang genome sequence. This study provides the statistical analysis of the sequence reads. Based on this information, further analysis on gene expression patterns influenced by Foc race infection within the tested groups and time points will help in the understanding of the host pathogenic responses. In future discovery on many new genes for diagnosis of plant infection could be achieved through excessive transcriptomic data

Standardized bioassays: an improved method for studying Fusarium oxysporum f. sp. cubense race 4 (FocR4) pathogen stress response in Musa acuminata cv. ‘Berangan’

To date, there is no standardized Fusarium bioassay protocol established owing partly to the wide variety of Fusarium oxysporum f. sp. cubense (Foc) isolates and banana cultivars present. Thus, validation of the infection parameters is deemed essential prior to each bioassay experiment. In the current study, a simple standardized workflow was developed based on available assays for testing Fusarium wilt disease response in Musa acuminata using M. acuminata cv. ‘Berangan’ of tissue-culture origin as a model. The phenotypic assays were able to detect external disease symptoms less than one week post-inoculation, while the molecular approach using RT-qPCR identified differential expression of catalase (CAT), pathogenesis-related 10 (PR10), phenylalanine ammonia-lyase (PAL) and xylanase (XYL) genes as early as day 0. The transcript levels of PR10 and XYL fluctuated over 4 days of Foc Race 4 (FocR4 C1 HIR isolate) infection while the expression of CAT steadily increased over time. In contrast, PAL was highly upregulated at 2 days post-inoculation. These signature changes suggest that all genes tested might be involved in the early defense response of ‘Berangan’ plants against FocR4 infection. ‘Berangan’ cultivar was found to be highly susceptible to Foc Race 4 (C1 HIR isolate) with leaf symptoms index (LSI) and rhizome discoloration index (RDI) scores of 4.257 and 5.971, respectively. The procedure elaborated in this study can be used as a reference Foc bioassay for reproducible and comparable results possibly across cultivars and test isolates due to its simple steps aided by integration of phenotypic and molecular approach

Vitamin E production in Amaranthus sp. and Allium porrum by co-suppression of homogentisate phytyltrransferase and tocopherol cyclase genes from Elaeis guineensis

Vitamin E is a fat-soluble vitamin that consists of four different tocopherol and tocotrienol isomers (α, β, γ and δ). Medical evidence suggests that vitamin E especially α-tocotrienol protects the cells against cancer, is a strong antioxidant and an effective neuroprotector. However, α-tocotrienol can only be found in seeds/cereal grain. In this study α-tocotrienol was produced by co-suppression of α-tocopherol by silencing the expression of two key vitamin E biosynthetic genes, homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) in Amaranthus sp. and Allium porrum leaves by transient transformation. HPT catalyses the condensation of homogentisate (HGA) and phytyl diphosphate (PDP). While TC forms the chromanol headgroup of the various tocopherol isomers. Gene silencing was performed using short sense conserved gene sequences of HPT and TC isolated from Elaeis guineensis. Through bioinformatics analysis, both isolated HPT and TC cDNAs were successfully verified to possess similar characteristics of the HPT and TC from other plant species. These isolated genes were predicted to encode a protein of 151 and 207 amino acid residues and they fell under the UbiA superfamily and tocopherol_cyc1 superfamily, respectively. Five gene constructs were generated p5b5, p5d9, p5f7, p4a11 and p4c9 driven either by the maize ubiquitin promoter (Ubi1P) or the Elaeis guineensis leaf-specific promoter (LHCB), in pDRB6b expression vector. All five recombinant vector constructs were successfully cloned, giving the expected size bands in gel electrophoresis analysis. The PCR product of the recombinant vector shared 78% identity with HPT from Zea mays (NM001112407.1). While the TC cDNA showed 79% identity with Vitis vinifera (XM002281388.2). In addition, plasmid stability in the transformed AGL1 showed stability ranging from 40% to 60%, identical to pDRB6b vector in AGL1. Optimised HPLC analysis showed that α-tocopherol was suppressed with different trend among the constructs (from minimum 4% up to 100%) in all transiently transformed plants. The p5f7 construct (LHCB-Ubi1intron-HPT-NosT) worked best in Allium porrum to suppress α-tocopherol in the range 62%-86%. While, p4c9 construct (LHCBTC-NosT) showed suppression up to 100% in Amaranthus sp. The results confirmed that the usage of LHCB showed better silencing than UbiP. In addition, with the usage of HPT gene, α-tocopherol was suppressed better (up to 86%) in Allium porrum than in Amaranthus sp. (75%). However, with TC gene the suppression was better in Amaranthus sp. (100%) than Allium porrum (57%). The production of α-tocotrienol was observed upon suppression of α-tocopherol using all recombinant vector constructs. With HPT gene the level of α-tocotrienol production was not dependent but with TC gene it was dependent on the level of α-tocopherol suppression. Overall, α-tocotrienol was produced more in Allium porrum than in Amaranthus sp. with p5d9 construct (Ubi1P-Ubi1intron-HPT-NosT). Thus, Amaranthus sp. and Allium porrum which do not accumulate α-tocotrienol naturally had shown to produce α-tocotrienol after suppression in the α-tocopherol production through transgenic manipulation. Finally, Agrobacterium -mediated transformation of inflorescence of Amaranthus sp. was carried out through drop by drop technique for producing transformed seeds. Nineteen transgenic plants were successfully obtained with transformation efficiency from 0.1% up to 2% for each constructs. This could serve as a potential system to engineer production of α-tocotrienol in transgenic Amaranthus sp

Female reproductive system of Amaranthus as the target for Agrobacterium-mediated transformation

Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods

Rt-qPCR profiling of pathogenesis related genes in Musa acuminata cv. ‘berangan’ seedlings challenged with Fusarium oxysporum F. SP. cubense tropical race 4

The expression profile of pathogenesis related genes are signatures of an infection response in plant cells. Pathogenic infections can increase or reduce gene expression in a plant system in a relatively specific pattern. These expression patterns can be used as standards in pathogenicity studies and, where phenotypic expression is normally used to gauge a plants response to infection, it could additionally present a more rapid and early screening reference tool. Three genes: catalase (CAT), pathogen related protein (PR10), and phenylalanine ammonia (PAL) all implicitly implicated in the plant disease response pathway were targeted for analysis during the infection of Fusarium oxysporum f. sp. cubense tropical race 4 (FOCR4) in banana Musa acuminata cv. Berangan seedlings after a standard challenge under growth room conditions. Distinct patterns of gene expression were observed at three infection time points by real time expression analysis. There was a sequential 10-fold reduction in expression for the PR gene while, the PAL and CAT genes were both upregulated. These results present a set of reference genes that could be used for screening of a plant’s response to Fusarium before the onset of symptoms. © 2019, University of Agriculture. All rights reserved

RNA-seq and ChIP-seq as complementary approaches for comprehension of plant transcriptional regulatory mechanism

The availability of data produced from various sequencing platforms offer the possibility to answer complex questions in plant research. However, drawbacks can arise when there are gaps in the information generated, and complementary platforms are essential to obtain more comprehensive data sets relating to specific biological process, such as responses to environmental perturbations in plant systems. The investigation of transcriptional regulation raises different challenges, particularly in associating differentially expressed transcription factors with their downstream responsive genes. In this paper, we discuss the integration of transcriptional factor studies through RNA sequencing (RNA-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq). We show how the data from ChIP-seq can strengthen information generated from RNA-seq in elucidating gene regulatory mechanisms. In particular, we discuss how integration of ChIP-seq and RNA-seq data can help to unravel transcriptional regulatory networks. This review discusses recent advances in methods for studying transcriptional regulation using these two methods. It also provides guidelines for making choices in selecting specific protocols in RNA-seq pipelines for genome-wide analysis to achieve more detailed characterization of specific transcription regulatory pathways via ChIP-seq

Evaluation of pathogenicity level of three different strains of fusarium oxysporum in Musa acuminata cv. Berangan

Fusarium oxysporum is one of the most adaptive and pathogenic fungus that causes wilting in banana. It is regarded as one of the economically destructive fungus and control of this disease is always ineffective once the disease becomes established in banana plantations. Various researches have been carried out, but sustainable solution is yet to achieve. In this study, we identified the infection phenotypically through LSI and RDI which can be only done after 5 th -week of post inoculation. With qPCR application, we identified the infection level much faster within 2 to 96 hrs. Disease severity index obtained from LSI and RDI value showed that Berangan is highly susceptible towards C1 HIR, 9888 and 2296. Meanwhile, qPCR analysis besides showing infection pattern as early as 2 hrs in Berangan was infected with C1 HIR, 9888 and 2296. We also found that additional information such as specific genes were expressed in the Berangan infected with C1 HIR, 9888 or 2296. This information can be used to identify the fusarium strain that is actually infecting Berangan. This could help planters to identify early infection of fusarium as well as strain recognition. © 2019, World Research Association. All rights reserved

Evaluation of pathogenicity level of three different strains of Fusarium oxysporum in Musa acuminata cv. Berangan

Fusarium oxysporum is one of the most adaptive and pathogenic fungus that causes wilting in banana. It is regarded as one of the economically destructive fungus and control of this disease is always ineffective once the disease becomes established in banana plantations. Various researches have been carried out, but sustainable solution is yet to achieve. In this study, we identified the infection phenotypically through LSI and RDI which can be only done after 5th-week of post inoculation. With qPCR application, we identified the infection level much faster within 2 to 96 hrs. Disease severity index obtained from LSI and RDI value showed that Berangan is highly susceptible towards C1 HIR, 9888 and 2296. Meanwhile, qPCR analysis besides showing infection pattern as early as 2 hrs in Berangan was infected with C1 HIR, 9888 and 2296. We also found that additional information such as specific genes were expressed in the Berangan infected with C1 HIR, 9888 or 2296. This information can be used to identify the fusarium strain that is actually infecting Berangan. This could help planters to identify early infection of fusarium as well as strain recognition

Induced production of α-tocotrienol by co-suppression of tocopherol cyclase gene in vegetable crops

This study involves the induced production of α-tocotrienol by the co-suppression of tocopherol cyclase gene. The Tocopherol/tocotrienol Cyclase (TC) is a key enzyme involved in the biosynthesis of α-tocotrienol. The partial conserved cDNA of TC gene isolated from the oil palm (Elaeis guineensis) that shared greater than 75% sequence identity with TC sequences from other plant species was used to silence TC gene expression in Amaranthus sp. and Allium porrum leaves by co-suppression strategies. This was carried out using recombinant vector constructs designated as p4a11 and p4c9 containing TC driven by the maize Ubiquitin promoter (Ubi1P) and the oil palm leaf-specific promoter (LHCB), respectively. In the Amaranthus sp. leaves infiltrated with p4a11, the α-tocopherol suppression was 85.0, 71.0 and 74.0% on day three, five and seven, respectively and complete suppression was observed in the leaves infiltrated with p4c9. Whereas, only 11.0, 7.0 and 29.0% suppression was detected in Allium porrum leaves infiltrated with p4a11 and 45.0, 4.0 and 57.0% of suppression was observed in Allium porrum infiltrated with p4c9 on day three, five and seven, respectively. Silencing of TC gene driven by LHCB (p4c9) causes the reduction of α-tocopherol production better than by Ubi1P (p4a11). The results have demonstrated that gene silencing through co-suppression is capable of producing α-tocotrienol. However, there is no correlation between the α-tocotrienol production and the α-tocopherol suppression. It has been suggested that the transgene mediated co-suppression mechanism had triggered the vitamin E biosynthesis metabolic pathway to shift towards producing α-tocotrienol