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Diuraphis noxia Kurdjumov (Russian wheat aphid, RWA) is considered a major agricultural pest to the wheat and barley industry, with losses in the USA exceeding US$1 billion attributed to this pest. The introduction of various resistant wheat cultivars in the latter part of the 1980s significantly reduced the impact this pest had on the industry. However, the recent development of new RWA biotypes in the USA and South Africa nullify the resistance of many existing wheat cultivars. Again the RWA became a serious threat to wheat production in these regions. The new RWA biotypes, however, present an opportunity to investigate biotype development and the mechanisms aphids utilize to overcome cultivar resistance. Wheat transcriptome studies, where different cultivars are infested by one or more RWA biotypes, could explain the mechanisms of the different modes of plant resistance against the RWA. Furthermore, these studies could also highlight which resistant pressures influences aphid biotype development.
Literature is reviewed in Chapter 2. Here the literature on the members and the bacteriumaphid and aphid-plant interactions is reviewed. The chapter begins with an introduction to the aphid, D. noxia, which includes details like its origin, taxonomy, morphology, biology, preferred hosts, economic impact, symptoms and control. This is followed by a brief introduction on the symbiotic relationship between bacterial endosymbionts and their role in aphid success. The plant host, Triticum aestivum, is reviewed before the biotic interaction, i.e. aphid-plant interaction, is introduced. The chapter concludes with the aphid-plant interaction. Here the physical interaction between the aphid and plant, including plant defence evading mechanisms with special focus on the aphid salivary enzymes, are discussed.
Diuraphis noxia (Aphididae: Macrosiphini)
Diuraphis noxia (Russian wheat aphid, RWA) resides within the tribe Macrosiphini of the subfamily Aphidinae (Heie 1992). It forms part of the phytophagous suborder Sternorrhyncha and can therefore be either in the Homoptera (together with the Auchenorrhyncha) or the Hemiptera (the Homoptera and Heteroptera) (Miles 1999).
Origin
The RWA is palaearctic in origin, i.e. central Asia to the Middle East. However, various introductions have resulted in a worldwide distribution to all arid and semi-arid cereal producing regions, with the only exception being Australasia (Hewitt et al. 1984; Du Toit 1986; 1987; Zemetra et al. 1990; Souza et al. 1991; Gonzalez et al. 1992; Blackman & Eastop 2000; Stray 2000; Stary & Lukasova 2002; Baker et al. 2003; Haley et al. 2004). In the Republic of South Africa, it was first detected in 1978 with major yield losses resulting in subsequent years (Du Toit & Walters 1984; Du Toit 1987).
Buchnera aphidicola sequence variation amongst biotypes
The leucine plasmid, pleuABCD and parts of the ptrpEG plasmid, 16S rDNA and trpB,
were amplified and sequenced (ABI BigDye v3.1. System, Applied Biosystems, USA (Table Appx 3.1). Fragments were aligned using ContigExpress (Vector NTI Advance 9, Invitrogen, USA) (Lu & Moriyama 2004), with cloned fragments first vector clipped using VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/). Inconsistencies in assemblies were manually investigated on the corresponding chromatograms and resolved with further sequencing. All BLAST analyses (Altschul et al. 1990; Altschul et al. 1997) were against the non-redundant Genbank database (NCBI, http://www.ncbi.nlm.nih.gov/).
Structural analysis
Bacterial promoters on the leucine plasmid were predicted with BPROM (http://softberry.com). Quickfold (Markham & Zuker 2005) and the Kinefold server (Xayaphoummine et al. 2005), using simulations of stochastic folding pathways from different random seed events, were used to predict the free energy values of the inverted repeat region upstream of leuA. The plasmids were screened for Rho independentterminators using FindTerm (http://softberry.com).
DNA and RNA extraction
Aphid total DNA was extracted using the DNAzol extraction protocol (Molecular Research Centre, Cincinnati, USA) and cleaned with the DNeasy cleanup kit (Qiagen, USA) that included the on column RNase treatment (Qiagen). All samples were quantified using the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, RSA). RNA extractions were performed on a 100 individuals collected with a soft brush and immediately frozen with liquid nitrogen. All extractions were done in accordance to the manufactures‘ protocols using the RNeasy Mini Kit (Qiagen) which included the on column DNase I treatment (RNase-free DNase set, Qiagen), before spectrophotometric quantification.
RT-qPCR
Relative gene expression levels of two genes on the pleuABCD plasmid of B. aphidicola, leuA and leuB, were quantified to assess whether the presence of the CCC-insert had any effect on the expression of these genes (Figure 4.3). A difference found in the expression levels has the potential to contribute to RWA adaptation to new hosts via the endosymbiont. It is known that B. aphidicola of the South African RWA biotypes have lower plasmid copy numbers (0.35 copies/bacterial chromosome) than their US counterparts (1.04 and 0.88 copies/bacterial chromosome for RWA-US1 and RWA-US2 respectively) (Chapter 3: Figure 3.4, Swanevelder et al. 2010). Significantly higher expression levels were obtained for leuA and leuB after normalization with rpoB (Figure 4.3) and 16S rRNA (not shown) in the South African B. aphidicola accessions with the CCC-insert than B. aphidicola without the insert (i.e. RWA-US1 and RWA-US2).
Conclusion
The initial transfer of the leucine biosynthetic genes, from the chromosome to a plasmid, was probably one of the main reasons for a successful aphid-Buchnera symbiosis (Latorre et al. 2005). However, different aphids require different levels of this essential amino acid. Indeed, the RWA has the ability to increase certain essential amino acids, including leucine, in susceptible hosts‘ phloem, but cannot achieve the same in a resistant host (Telang et al. 1999; Porter & Webster 2000; Sandstrom et al. 2000; Ni et al. 2001). Previously it was believed that the fine tuning of leucine production in an aphid species was done through changes in the plasmid copy number (Thao et al.1998; Plague et al. 2003; Latorre et al. 2005). However, the increase in the leuA-leuB ORF transcripts relative to the known plasmid copy numbers, suggest that this could be a form of regulation within the species. The existence of variable regions within an aphid species and differences in structural stabilities of either the plasmid or leader sequences within Buchnera plasmid could arguably support a regulatory mechanism for leucine control. The fact that the same insertion occurred twice, independently and involved multiple nucleotides that are not part of the Buchnera‘s genome bias (AT-rich genome) (Chapter 3; Swanevelder et al. 2010), further supports this hypothesis. We therefore propose that the variation within the inverted repeat region, together with plasmid copy numbers, are used by Buchnera to control gene expression, either through higher expression levels or via 5‘ UTR mRNA stabilization.
CHAPTER 1 INTRODUCTION CHAPTER 2 APHID-PLANT-ENDOSYMBIONT INTERACTION: THE RUSSIAN WHEAT APHID, ITS HOSTS AND ENDOSYMBIONT, BUCHNERA APHIDICOLA DIURAPHIS NOXIA (APHIDIDAE: MACROSIPHINI) Origin
Description
Biology
Genetics
Biotypes
Hosts
Symptoms
Economic losses
Control
CHAPTER 3 LIMITED ENDOSYMBIONT VARIATION IN DIURAPHIS NOXIA (HEMIPTERA: APHIDIDAE) BIOTYPES FROM THE USA AND SOUTH AFRICA
INTRODUCTION
MATERIALS AND METHODS
Diuraphis species and biotypes
Biotypic endosymbiont investigation
Buchnera aphidicola sequence variation amongst biotypes
Structural analysis
Plasmid copy numbers SYMBIOSIS: VARIATION IN BUCHNERA APHIDICOLA’S LEUCINE PLASMID MAY CONFER ADVANTAGE TO RUSSIAN
WHEAT APHID BIOTYPE
INTRODUCTION
MATERIALS AND METHODS
Aphids
DNA and RNA extraction
Leader sequence determination
The inverted repeat region in the Aphididae
Software analysis
CHAPTER 5 JUST HOW DO AFFYMETRIX NORMALIZATION METHODS COMPARE? STATISTICS CONTEMPLATE BIOLOGY
INTRODUCTION
MATERIALS AND METHODS
Experimental design
Aphids and plant material
Data analysis
RESULTS
Quality control of slides, background correction and normalization
CHAPTER 6 CONCLUSION
