Agriculture Reference
In-Depth Information
isolates and are non-functional alleles of the avirulence gene, producing variants of
NIP1 which are not detected by
Rrs1
resistant plants. No single feature of the
NIP1
sequence distinguishes these alleles from those in avirulent isolates and the proposed
causes of their non-functionality are different: alleles 3 and 4 produce ineffective
NIP1 elicitor proteins (Fiegen and Knogge, 2002) and allele 8 may also be
ineffective, whereas NIP1 may not be expressed in isolates with alleles 7 and 17. In
addition to mutations in the
NIP1
sequence, deletions of
NIP1
were found in other
virulent isolates of
R. secalis
and in fact were much more frequent than the mutant,
non-functional alleles.
(b) Use of gene sequences in population studies
Sequences of alleles of an avirulence gene may be used in population genetic studies
of a pathogen. For example, Schürch
et al.
(2004) estimated the gene diversity of
NIP1
in populations from four continents. DNA sequences have the potential to be
used as a replacement or a supplement to pathology tests because well-designed
PCR tests of sequence variation are now relatively quick and use techniques which
are standard in any molecular genetics laboratory. Pathology tests of virulence, by
contrast, may take several weeks and require highly specialised skills.
However, if sequences are to be used widely in pathogen surveys, several
potential obstacles need to be avoided, the first three of which may be illustrated by
reference to the work of Schürch
et al.
(2004). The source of most of them is the
large effective population size of most plant pathogen species, which allows many
alleles of any one gene to be maintained in the population. The first is that no single
nucleotide base may distinguish precisely between avirulence and virulence. A
second issue, which arises from the first, is that when a new allele is detected,
pathology tests are essential to determine whether it encodes a functional avirulence
allele or not. Thirdly, the testing procedure must be sufficiently reliable that the
experimenter can be sure that null PCR amplifications result from the absence of the
gene (or at least the part of the gene encoding the primer hybridisation sites) and not
merely from a poor sample. Fourthly, an efficient PCR diagnostic test requires
amplification of a DNA fragment by a single pair of primers. Any sequence
variation outside the amplified region that contributes to phenotypic variation will
not be detected. Lastly, virulence may result from non-expression of the avirulence
gene, which might be caused by variation in a different gene that interacts with the
avirulence gene.
Despite these scientific and technical challenges, it is likely that cloned avirulence
genes will begin to be used in pathogen surveys and that their use in population
genetic research will increase. If the aim is to estimate frequencies of virulent and
avirulent phenotypes of the pathogen in target populations, misidentification of
occasional isolates may not matter very much. If, however, accurate identification of
individual isolates is essential, the 'gold standard' remains the pathology test but
sequence data may provide useful supporting information.
