Maize Streak Virus Research Group at the University of Cape Town

Introduction

As mentioned previously, maize streak virus is one of the most severe threats to the maize growing community in Africa, because of its potential to cause crop failures or yield reductions.  We decided to concentrate our efforts on this virus as long ago as 1984 - coincidentally, just about when the first two sequences came out, to our not inconsiderable dismay - because:

1. it was localised to Africa and neighbouring states, which gave us a geographical advantage in working on it;
2. it was a potentially severe pathogen (and we might get money for it...);
3. Ed Rybicki was sick of working on brome mosaic virus.

From small beginnings, we have managed to build up a quite respectable body of work, and a dynamic and exciting research area.  This page will explore the history of MSV research in our Department, and will describe current research efforts.  Links to other labs will also be provided.

Lab History

The work was inherited from Assoc Prof M Barbara von Wechmar, who - with the late Dr I Harpaz, from Israel - had started our non-viruliferous Cicadulina mbila colony in 1978 (with hoppers from Dr GDJ "Org" van Rensburg, presently of the Grain Crops Institute).  Barbara had previously done some of the only virological work on the virus in this country in modern times (von Wechmar, PhD Thesis, 1968).  Bob Milne - presently at the Istituto di Fitovirologia Applicata del CNR in Torino - took the first EM photos of our virus, also in 1978, while on sabbatical here.   These were later added to by Kassie Kasdorf, then with the Plant Protection Research Institute in Stellenbosch.  Barbara and Bob co-authored a paper on the extraction of the virus (von Wechmar and Milne, 1983), and Barbara and the hugely inventive Dr Alfred Polson published on a technique called "electroinfection" - basically, using a 9V battery to electrophorese virus particles into plants - to infect maize plants with MSV (Polson and von Wechmar, 1980).  Although Barbara continued to collect specimens of MSV around the country, and to maintain the hopper colony, she had largely shelved the academic side of things until we started the new initiative.

Beverley Clarke - MSc thesis 1987 - started working on the virus in 1985: her project was to characterise as fully as possible as many isolates / strains of the virus as possible, with a view to determining the range of genotypes of the virus in South Africa and neighbouring territories.  She pioneered a purification protocol for dsRF-DNA that we still use in modified form today: "if it looks like a plasmid, purify it like a plasmid" - so she did, using solutions made for E coli minipreps - and it worked wonderfully.  She restriction mapped three viruses after cloning full-length genomes in E coli, as well as doing some serology on them (Clarke et al. 1989), and provided the ammunition for Ralph Kirby - our internal collaborator and DNA methods guru - to do the first phylogenetic analysis on map sites (Kirby et al., 1989).  This work provided an excellent foundation for subsequent efforts, and is discussed here as well.

Fiona Hughes started a PhD project in the lab in 1987, again, with the object of characterising the diversity of the virus.  She refined the art of cloning and restriction mapping of virus isolates (see Hughes et al., 1992), and did a good deal of differential hybridisation as well: this method was used to identify distinctly different viruses, which were later characterised as panicum streak virus (PanSV-Karino) and sugarcane streak virus (SSV-Natal and -Mauritius), as well as distinct strains of the virus (MSV-Setaria, from a Setaria sp. from a sugarcane field, and MSV-WES, from an Eleusine sp. and from a wheat from the Free State Province of South Africa).  She also pioneered the concept of using restriction fragment length polymorphisms (RFLPs) to study virus diversity from field-sampled plants and different genomes cloned from the same plant: this is discussed here.  Fiona went on to partially sequence a PCR-generated fragment of SSV-Mauritius (Rybicki and Hughes, 1990), and to completely sequence an unfortunately non-infectious clone of SSV-Natal (Hughes et al., 1993). Fiona got her PhD in 1991, and went on to do two post-doc stints in the lab, interrupted and then terminated by successive babies - Julian and Lucy.  She continued her diversity work, and also incidentally made the first agroinfectious MSV clone from Africa: MSV-Komatipoort, a moderately severe maize isolate of MSV from the Mpumalanga Province of S Africa.  The clone was subsequently sequenced by Mrs Di "Deoxy" James from clones provided by Fiona. 

Fiona also started what is now a significant thrust in our lab, by constructing various MSV-derived Rep antisense expression plasmids for the engineering of transgenic resistance into maize: these were biolistically transformed into embryogenic callus by Bill Gordon-Kamm (then at De Kalb, Inc) in the USA.   Unfortunately, even though we could later regenerate plants from the callus, these were not noticeably MSV resistant. 

This work marked the entry of Prof Jennifer Thomson (Head, Dept Microbiology) into the MSV-related research area; first as a student co-supervisor (Michelle Babaya, MSc 1988; see below), then as Fiona's co-employer, and since as a co-supervisor and major collaborator.   She and Ed Rybicki have been instrumental in setting up a plant tissue culture "unit" - complete with Du Pont helium "gun" - primarily for the genetic engineering of maize.  Sandy Lennox was employed as a research officer to establish maize tissue culture techniques, and has succeeded in establishing protocols for the reliable regeneration of plants from transformed HiII callus, and for the reliable regeration of a number of local maize breeding lines and other material. 

In the meantime, Barbara von Wechmar had continued to work on biological aspects of MSV: she established the concept of a "differential panel" of local breeding lines and varieties of maize, as well as of wheat, barley and some grasses, for the fine differentiation of leafhopper-transmitted virus isolates and strains by symptomatology and host range (von Wechmar and Hughes, 1990).  We have since expanded considerably on this concept (see here, and below); however, this was the first comprehensive use of local cereal varieties in modern times, with well-characterised viruses, to differentiate large numbers of virus isolates.

A number of Honours students and others also worked on MSV in the years between 1985 and 1993.  Michelle Babaya - co-supervised first with Prof Dave Woods, then with Prof Jenny Thomson - cloned and probably expressed maize streak virus genes in tobacco (Babaya, MSc Thesis, 1990).  Ann Pithey managed to clone and sequence, and express as a beta-galactosidase fusion protein, the movement protein of an MSV isolate from Port Elizabeth (MSV-PE) (Pithey, Hons Thesis, 1988).  Erik Edge carried on the RFLP approach to the investigation of the diversity of MSV in single-field samples, discussed in more detail  here (Edge, Hons Thesis, 1989). 

Ed Rybicki pioneered the use of PCR in the lab, with a set of degenerate "Mastrevirus-specific" oligonucleotide primers which could be used to amplify a 250-odd base pair sequence from the RepB region of just about any grass-infecting Mastrevirus (Rybicki and Hughes, 1990; Rybicki et al., 1998).   Susan Dennis pioneered direct PCR fragment sequencing in the lab, and sequenced a number of PCR-generated products, including from MSV-WES and MSV-Set, while working as a technician up to 1991.  David Wallace (Wallace, Hons Thesis, 1990) studied Zimbabwean isolates of MSV from around Harare obtained from Kent Short, then at CIMMYT, by PCR and direct sequencing, using type-specific primers.  He also showed it was possible to directly sequence plasmid-like dsRF-DNA extracted from infected maize plants, using virus-specific primers.  Grant Napier carried on with sequencing PCR-generated products, this time from cloned fragments, generated using group-specific primers (Napier, Hons Thesis 1992).  Aspects of this work were published in Molecular Plant Pathology On-Line.

Mark Fyvie managed to completely sequence an infectious clone of panicum streak virus strain Karino (PanSV-Kar), originally cloned by Fiona Hughes, and made agroinfectious by Wendelin "Popeye" Schnippenkoetter (Fyvie, Hons Thesis 1993; Schnippenkoetter, PhD Thesis, 1998).  Harvey Jones worked on Zimbabwean isolates of MSV, and managed to clone, map, and partially sequence several similar viruses (Jones, Hons Thesis, 1994).

The present era in the lab - with more than one or two students working on the projects at the same time - was partly made possible by Foundation for Research Development (now the National Research Foundation, South Africa) funding to Ed Rybicki, which started in a significant way in 1993.   Ed was able to add more students to the single PhD student working at the time, and significantly accelerate our progress.  Jennifer has also received FRD funding, as well as monies from African Explosives and Chemical Industries, Ltd.,  the Claude Harris Leon Foundation, and Pannar Ltd.   Ed Rybicki essentially supervises molecular virological projects on MSV;   Jennifer and Ed co-supervise biotechnological / genetic engineering projects using MSV, and Jennifer supervises other plant genetic engineering projects.

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