Noëlani van den Berg

Elicitor and Fusarium-induced expression of NPR1-like genes in banana.

The non-expressor of pathogenesis-related genes 1 (NPR1) is an essential positive regulator of salicylic acid (SA)-induced pathogenesis-related (PR) gene expression and systemic acquired resistance (SAR). Two novel full length NPR1-like genes; MNPR1A and MNPR1B, were isolated from banana by application of the PCR and rapid amplification of cDNA ends (RACE) techniques. The two identified MNPR1 sequences differed greatly in their expression profile using quantitative real time (qRT)-PCR following either elicitor or Fusarium oxysporum Schlecht f. sp. cubense (Smith) Snyd (Foc) treatment. MNPR1A was greatly expressed after Foc treatment with higher and earlier expression in the Foc-tolerant cultivar GCTCV-218 than in the sensitive cultivar Grand Naine. In comparison, MNPR1B was highly responsive to SA, but not to methyl jasmonate (MeJA) treatment, in both the tolerant banana cultivar GCTCV-218 and the more sensitive cultivar Grand Naine. Expression of the MNPR1 genes further directly related to PR gene expression known to be involved in fungal resistance. Reduced sensitivity to Foc in GCTCV-218 might be partially attributed to the higher and an earlier expression of both MNPR1A and PR-1 in this cultivar after Foc treatment. Further characterisation of the MNPR1 genes through complementation of Arabidopsis npr1 mutants and overexpression studies in banana cultivars is the subject of ongoing and future work.

High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

EST sequencing and gene expression profiling of defence-related genes from Persea americana infected with Phytophthora cinnamomi

BackgroundAvocado (Persea americana) belongs to the Lauraceae family and is an important commercial fruit crop in over 50 countries. The most serious pathogen affecting avocado production is Phytophthora cinnamomi which causes Phytophthora root rot (PRR). Root pathogens such as P. cinnamomi and their interactions with hosts are poorly understood and despite the importance of both the avocado crop and the effect Phytophthora has on its cultivation, there is a lack of molecular knowledge underpinning our understanding of defence strategies against the pathogen. In order to initiate a better understanding of host-specific defence we have generated EST data using 454 pyrosequencing and profiled nine defence-related genes from Pc-infected avocado roots.Results2.0 Mb of data was generated consisting of ~10,000 reads on a single lane of the GS FLX platform. Using the Newbler assembler 371 contigs were assembled, of which 367 are novel for Persea americana. Genes were classified according to Gene Ontology terms. In addition to identifying root-specific ESTs we were also able to identify and quantify the expression of nine defence-related genes that were differentially regulated in response to P. cinnamomi. Genes such as metallothionein, thaumatin and the pathogenesis related PsemI, mlo and profilin were found to be differentially regulated.ConclusionsThis is the first study in elucidating the avocado root transcriptome as well as identifying defence responses of avocado roots to the root pathogen P. cinnamomi. Our data is currently the only EST data that has been generated for avocado rootstocks, and the ESTs identified in this study have already been useful in identifying defence-related genes as well as providing gene information for other studies looking at processes such as ROS regulation as well as hypoxia in avocado roots. Our EST data will aid in the elucidation of the avocado transcriptome and identification of markers for improved rootstock breeding and screening. The characterization of the avocado transcriptome will furthermore form a basis for functional genomics of basal angiosperms.

De Novo sequencing, assembly, and analysis of the root transcriptome of Persea americana (Mill.) in response to Phytophthora cinnamomi and flooding

Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.

Dual RNA-Sequencing of Eucalyptus nitens during Phytophthora cinnamomi Challenge Reveals Pathogen and Host Factors Influencing Compatibility.

Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction.

Phylogenetic and expression analysis of the NPR1-like gene family from Persea americana (Mill.)

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) forms an integral part of the salicylic acid (SA) pathway in plants and is involved in cross-talk between the SA and jasmonic acid/ethylene (JA/ET) pathways. Therefore, NPR1 is essential to the effective response of plants to pathogens. Avocado (Persea americana) is a commercially important crop worldwide. Significant losses in production result from Phytophthora root rot, caused by the hemibiotroph, Phytophthora cinnamomi. This oomycete infects the feeder roots of avocado trees leading to an overall decline in health and eventual death. The interaction between avocado and P. cinnamomi is poorly understood and as such limited control strategies exist. Thus uncovering the role of NPR1 in avocado could provide novel insights into the avocado – P. cinnamomi interaction. A total of five NPR1-like sequences were identified. These sequences were annotated using FGENESH and a maximum-likelihood tree was constructed using 34 NPR1-like protein sequences from other plant species. The conserved protein domains and functional motifs of these sequences were predicted. Reverse transcription quantitative PCR was used to analyze the expression of the five NPR1-like sequences in the roots of avocado after treatment with salicylic and jasmonic acid, P. cinnamomi infection, across different tissues and in P. cinnamomi infected tolerant and susceptible rootstocks. Of the five NPR1-like sequences three have strong support for a defensive role while two are most likely involved in development. Significant differences in the expression profiles of these five NPR1-like genes were observed, assisting in functional classification. Understanding the interaction of avocado and P. cinnamomi is essential to developing new control strategies. This work enables further classification of these genes by means of functional annotation and is a crucial step in understanding the role of NPR1 during P. cinnamomi infection.

Putative pathogenicity genes of Phytophthora cinnamomi identified via RNA-Seq analysis of pre-infection structures

Phytophthora cinnamomi is an economically important oomycete that infects more than 3,000 plant species. We aimed to identify the repertoire of genes expressed during pre-infection stages by analysing an RNA-Seq library of cysts and germinating cysts of a P. cinnamomi isolate, originating from Persea americana. Over 70,000 transcripts were identified from 225,049 contigs, assembled from 13 million Illumina paired-end reads. Contaminant sequences were eliminated, resulting in 37,534 transcripts used in further analysis. A total of 1394 transcripts had a putative role in pathogenesis. Genes aiding in detoxification and metabolite transport (cytochrome P450 and ABC transporters) and protection against oxidative stress were most abundant, followed by the genes coding cell wall degrading enzymes. The transcript set included 44 putative RXLR effector genes and genes encoding elicitin and necrosis-inducing proteins. Expression patterns of seven putative pathogenicity genes (encoding RXLR-, necrosis-inducing Phytophthora protein 1 (NPP1), elicitin, polygalacturonase, cellulose binding and elicitor lectin (CBEL), mucin, and adhesion proteins) were assessed across four in vitro developmental stages of P. cinnamomi. High expression of these genes in zoospores suggests their functional importance in the subsequent developmental stage, germination of cysts, implying a role in pre-infection. This work is the first step towards understanding the molecular basis of infection strategies employed by P. cinnamomi.

Transcriptome analysis of an incompatible Persea americana-Phytophthora cinnamomi interaction reveals the involvement of SA- and JA-pathways in a successful defense response

Phytophthora cinnamomi Rands (Pc) is a hemibiotrophic oomycete and the causal agent of Phytophthora root rot (PRR) of the commercially important fruit crop avocado (Persea americana Mill.). Plant defense against pathogens is modulated by phytohormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid. The role of specific signaling pathways induced and regulated during hemibiotroph-plant interactions has been widely debated. Some studies report SA mediated defense while others hypothesize that JA responses restrict the spread of pathogens. This study aimed to identify the role of SA- and JA- associated genes in the defense strategy of a resistant avocado rootstock, Dusa in response to Pc infection. Transcripts associated with SA-mediated defense pathways and lignin biosynthesis were upregulated at 6 hours post-inoculation (hpi). Results suggest that auxin, reactive oxygen species (ROS) and Ca2+ signaling was also important during this early time point, while JA signaling was absent. Both SA and JA defense responses were shown to play a role during defense at 18 hpi. Induction of genes associated with ROS detoxification and cell wall digestion (β-1-3-glucanase) was also observed. Most genes induced at 24 hpi were linked to JA responses. Other processes at play in avocado at 24 hpi include cell wall strengthening, the formation of phenolics and induction of arabinogalactan, a gene linked to Pc zoospore immobility. This study represents the first transcriptome wide analysis of a resistant avocado rootstock treated with SA and JA compared to Pc infection. The results provide evidence of a biphasic defense response against the hemibiotroph, which initially involves SA-mediated gene expression followed by the enrichment of JA-mediated defense from 18 to 24 hpi. Genes and molecular pathways linked to Pc resistance are highlighted and may serve as future targets for manipulation in the development of PRR resistant avocado rootstocks.