In July 2000, the finger of blame for a mysterious mass killer of Californian oak trees
came to rest on a previously undescribed plant pathogen. From the initial identification of
Phytophthora ramorum , it took less than four years to produce a
draft sequence of its genome, one of the fastest-ever discovery-to-sequence stories for a
complex pathogen. This achievement was a United States initiative, facilitated by the
injection of federal and state funding into
Phytophthora research. But the US is not alone in the battle
against this genus.
Phytophthora species cause thousands of millions of dollars of
damage to the world's commercial crops every year: they blight potatoes and tomatoes,
devastate the lucrative soybean, and rot cacao, threatening the world's supply of chocolate
(Figure 1). But for Sophien Kamoun, Associate Professor of Plant Pathology at Ohio State
University (Wooster, Ohio, United States), these destructive organisms present an exciting
opportunity. There are some 60 species of
Phytophthora , but so little is known about the genus, he says,
that there are things about its species we didn't even know we didn't know. ‘What I find
really exciting,’ he says, ‘is discovering these unknown unknowns.’
The most infamous of the
Phytophthora pathogens is the potato late blight,
P. infestans . It was this species that led to the Irish potato
famine in the mid-1840s, which resulted in the death or displacement of millions. Today,
P. infestans is estimated to cost potato and tomato farmers
US$5,000,000,000 a year in lost revenue. The story for the soybean pathogen
P. sojae is similar, causing loss of more than US$1,000,000,000
a year to soybean growers. In addition to the direct economic impact of these pathogens,
introduced
Phytophthora can cause severe damage to native flora. The most
recent
Phytophthora on the scene is
P. ramorum , which has caused ‘sudden oak death’ (SOD) in tens
of thousands of oak trees across the coastal counties of California, is now present in at
least three other US states and is threatening to take on the native flora of the entire
North American continent. It is also lurking in Europe, although apparently with less
devastating consequences.
Molecular Machinery
With this kind of impact, it's no surprise that money has poured into research on
Phytophthora . This year, the US federal government will channel
US$7,400,000 toward research into SOD. A major focus of this funding is genomics.
Sequencing the genomes of several
Phytophthora will help clarify the phylogeny and evolution of
these enigmatic organisms (Box 1) and improve methods of detection and identification.
Ultimately, however, sequencing should reveal the molecular tricks that this genus uses to
subvert the defences of its plant hosts, allowing scientists to come up with new ways to
combat these troublesome organisms.
The
P. infestans sequencing initiative, coordinated by Kamoun, has
recently completed a survey sequence of the genome that gives an initial understanding of
how this organism is structured. Perhaps most striking is its size. ‘It's a huge genome,’
says Kamoun. At about 250 megabases (Mb), it's about twice the size of the
Arabidopsis genome. His latest research, published in the
Journal of
Biological Chemistry (Tian et al. 2004), describes a
P. infestans protease inhibitor—extracellular protease inhibitor
1 (EPI1)—that could be one of a unique class of suppressor proteins that
Phytophthora deploy to infect and counteract host defences. The
pathogen seems to upregulate the
epi1 gene during colonisation of its host. EPI1 inhibits plant apoplastic
proteases—extracellular enzymes that are part of the host's defensive armoury that have
evolved to prevent foreign proteins entering cells.
‘Based on its biological activity and expression pattern, EPI1 may function as a disease
effector molecule and may play an important role in
P. infestans colonisation of host apoplast,’ Kamoun and his
colleagues report (Tian et al. 2004). If further research confirms this function for EPI1,
then it will become one of just a handful of pathogen molecules that have been shown to
suppress host plant defenses. A search in sequence databases for matching motifs reveals
just one similar sequence in the entire bacterial and fungal kingdoms. However,
apicomplexans like
Toxoplasma gondii that transit through the mammalian digestive
tract also appear to secrete protease inhibitors allied to EPI1. This similarity suggests
an analogy between plant apoplasts and mammalian digestive tracts. Both environments are
rich in proteases, but nevertheless are colonised by a variety of microbial pathogens. In
the case of an apoplast, the pathogen is
P. infestans , whilst in the mammalian gut, it's
T. gondii —and although they are phylogenetically distant, these
pathogens seem to have independently recruited similar secreted proteins to inhibit the
defensive proteases produced by their hosts. Interestingly, whilst
T. gondii inhibitors inhibit gut enzymes trypsin and
chymotrypsin, EPI1 does not, suggesting that coevolution between the inhibitors and their
target proteases may shape the specificity of these pathogenic enzymes.
Armed with this new insight into the molecular cunning of
P. infestans , Kamoun hopes that it will be possible to come up
with ways of slowing disease progression. Importantly, it looks likely that protease
inhibitors such as EPI1 are present in other
Phytophthora species. There are significant matches between the
epi1 gene sequence and motifs from at least five other closely related
species. So any methods of blocking the action of protease inhibitors in
P. infestans might also work against other
Phytophthora . The protease inhibitors are one of Kamoun's
‘unknown unknowns’. ‘It's an example of something that we had absolutely no idea was in the
genome,’ he says.
Even more advanced than the
P. infestans genome project is an ongoing collaboration between
the Virginia Bioinformatics Institute (Blacksburg, Virginia, United States) and the US
Department of Energy's Joint Genome Project based in Walnut Creek, California. The focus
here is the soybean pathogen
P. sojae and the SOD pathogen
P. ramorum , for which draft sequences are now complete
(www.jgi.doe.gov/). The genomes are much smaller than that of the 250-Mb
P. infestans —
P. sojae is about 90 Mb and
P. ramorum is just 55 Mb. This, in part, explains the
comparative speed of these sequencing efforts. But another factor is undeniably the fear of
the unknown
P. ramorum , which in 2002 netted the Virginia–California
initiative US$3,800,000 in federal funding to describe its genome. Ultimately, however, the
sequence of one species will help to inform on the sequence of other related species.
Brett Tyler, Research Professor at the Virginia Bioinformatics Institute, is focusing on
the molecular tools used by
P. sojae to infect its host. He agrees with Kamoun that
understanding this machinery is the way to devise new control measures that could give
plants the upper hand in the evolutionary arms race against their
Phytophthora pests. At present, most strategies to limit the
damage caused by species like
P. infestans and
P. sojae rely on developing resistant cultivars by selective
breeding of varieties with major resistance genes—single genes that can block a pathogen.
However,
Phytophthora seem able to find ways to overcome these efforts. ‘
P. infestans is absolutely notorious for its ability to
genetically change in response to a major resistance gene,’ says Tyler. ‘Typically major
resistance genes in potato barely last a single season.’
Things look better for the soybean. New cultivars containing major resistance genes show
resilience to
P. sojae for five to ten years. However, this resistance is
starting to break down, and breeders are running out of major resistance genes with which
to conjure new varieties. The alternative, says Tyler, is quantitative or multigenic
resistance, which relies on getting plants to express several resistance genes at once,
each of which makes a small contribution to the plant's overall resistance. It should be
much harder for
P. sojae to evolve a new attack against this kind of robust
defence. The search is also on for new genes that could be used to encourage quantitative
resistance to host species. One particularly promising approach is to pit pathogen against
host to see which genes are switched on. The host should upregulate genes that defend it
against infection and the pathogen should upregulate genes that it needs to attack. The
discovery that plants produce proteases and that
Phytophthora have responded by secreting protease inhibitors to
disable them is important if long-lasting solutions are to be found. ‘We just need to find
a way to introduce new protein-degrading enzymes into the plant that the pathogen doesn't
know how to block,’ says Tyler. ‘With these genomic tools we can really accelerate the pace
at which we can evaluate different possible protective measures.’
Epidemiology: Identifying the Culprit
An additional benefit of the abundance of genetic information is that species
identification is becoming increasingly sophisticated. At a glance,
Phytophthora can be mistaken for a fungus, so DNA profiling of
isolates is crucial if species and strains of species are to be identified correctly so
that action appropriate to each infection can be taken. Since 2000, Matteo Garbelotto,
Adjunct Professor of Mycology and Forest Pathology at the University of California at
Berkeley (Berkeley, California, United States), has spent a significant part of his working
life tracking the spread of
P. ramorum , the
Phytophthora that has killed off vast tracts of oak trees in
native Californian forest (Box 2).
DNA analysis has been crucial to confirm suspected cases of
P. ramorum , and has now revealed that infections have reached
at least three other US states. This spread is probably due to the movement of infected
ornamentals like rhododendron (
Rhododendron spp.) and viburnum (
Viburnum spp.), which seem to act as carriers for the pathogen.
‘What we're seeing is a parallel to what has been happening throughout Europe, where the
infection has basically moved using the commercial routes of the ornamental plant
industry,’ Garbelotto says.
P. ramorum in Europe
P. ramorum does not appear to have the same devastating
consequences in Europe as it does in the US—at least not yet. It's not entirely clear why,
but it could have something to do with the structure of the bark of different host species,
suggests Garbelotto. ‘We normally see more infection where we have more corrugation, and
that's because water accumulates in the fissures … where the zoospores have a chance to
infect the bark.’ However, he notes, European beech (
Fagus sylvatica ) appears to be extremely susceptible to
P. ramorum . ‘If it reaches areas where there are a lot of
beeches, it could potentially mirror what's happening in California,’ he warns.
In Europe, symptoms characteristic of
P. ramorum infection were first described on rhododendrons in
The Netherlands in 1993. Once this was confirmed to be the same species as the pathogen
responsible for Californian SOD, there was speculation that
P. ramorum had either been introduced to the US from Europe or
vice versa. However, in December 2002, it emerged that these two populations are of
different mating types—A1 in Europe and A2 in the US. The latest research from Garbelotto
and his colleagues, due to be published in
Mycological Research (Ivors et al. 2004), supports this
interpretation, demonstrating that although they belong to the same species, A1 and A2 are
distinct lineages and have not exchanged genes for a long time. But last year, a batch of
isolates from infected camellias (
Camellia spp.) and rhododendrons in a nursery in Washington
state showed that the A1 and A2 strains were living side by side. ‘That was a big surprise
for us,’ recalls Garbelotto. ‘We had no knowledge at that point that both the European and
the North American type could be present in the same nursery.’
The Threat of Recombination
Hybridisation between different species of
Phytophthora can produce a new species with different properties
from those of either parent. One of the best cases comes from Europe, where a new
Phytophthora emerged in 1993 that began to attack alder trees (
Alnus spp.). Research carried out by scientists in the United
Kingdom demonstrated that the alder
Phytophthora was a product of a hybridisation event between
P. cambivora and an unknown species similar to
P. fragariae , neither of which attacks alder.
Given this propensity for
Phytophthora species to hybridise and new phenotypes to emerge,
there is legitimate concern that sexual recombination between the A1 and A2 mating types
could produce something more devastating than either form. Within the controlled confines
of his laboratory, Garbelotto has been exploring whether the two types of
P. ramorum can get it together. Initial findings are that
oospores—the product of sexual recombination—are being produced, although most of them
abort before they reach maturity. However, 30% progress further, and (microscopically, at
least) look like they could be functional. ‘They'll germinate,’ he predicts.
The threat that hybridisation could create a novel strain with a different host range is
a concern that the UK is also taking seriously. In December 2003, an entirely new
Phytophthora was isolated from two sites in England. Although
the new species—currently referred to as
Phytophthora taxon C sp. nov. (
P . taxon C)— appears to cause relatively mild damage to its
beech and rhododendron hosts, the UK's Department for Environment, Food and Rural Affairs
(DEFRA) acknowledges that hybridisation of
P . taxon C with
P. ramorum could have serious consequences. ‘The potential for
the pathogen to adapt further to its putative new environment intrinsically or via
hybridisation is not known,’ note the authors of a DEFRA report on the mystery species
(www.defra.gov.uk/planth/pra/forest.pdf). ‘Long-distance spread could easily occur through
the movement of infected stock of rhododendron or beech and possibly other (as yet unknown)
hosts,’ they warn.
DEFRA is monitoring the situation closely (Figure 4). However, on the basis of a
preliminary DNA analysis,
P . taxon C and
P. ramorum are only distantly related, making hybridisation
unlikely, says Joan Webber, Head of Pathology at the UK government's Forestry Commission.
The closest known relative of
P . taxon C is
P. boehmeriae , a pathogen that has been recorded on several
species of tree in China and Australia, and on cotton in China and Greece, suggesting
possible origins for the newly described species. But, says Webber, the sequence match
between
P . taxon C and
P. boehmeriae is only 92%—not especially close. Much more
evidence is needed to build a strong case for the origin of this new
Phytophthora , she says.
Origins
Indeed, it has taken more than 150 years to track down the geographical origin of the
P. infestans strain that caused the Irish potato famine. Jean
Beagle Ristaino of North Carolina State University (Raleigh, North Carolina, United States)
is due to publish in
Mycological Research an analysis of DNA extracted from diseased
potato plants preserved from the nineteenth-century Irish epidemic (May and Ristaino 2004).
It had long been suspected that the famine was caused by the Ib strain of
P. infestans , which is presumed to have originated in Mexico.
However, Ristaino's molecular evidence spotlights the Ia strain and traces its probable
roots to the Andes. The infection could have found its way from South America to Europe and
the US via exports of potato seed on steamships, she speculates.
This kind of forensic treatment is more than just interesting. Tracing a
Phytophthora species to its site of origin could reveal what
keeps them at bay in the areas where they are native, and might suggest new ways to manage
them when they are introduced to a different ecosystem, says Garbelotto: ‘There's a huge
amount of information that can be learned from understanding where they're coming from.’ So
where do pathogens like
P. ramorum originate? The best lead, Garbelotto says, is the
ease with which it infects rhododendron. These ornamentals are natives of Asia, but there
are only a few places where the climate would suit
P. ramorum . The most promising, he suggests, are the Southern
Himalayas, the Tibetan plateau, or Yunnan province in China. But these are big places, and
Garbelotto has plenty on his plate in his battle against the Californian SOD. ‘I am not
very hopeful that we'll ever be able to find out where it comes from,’ he says.
