By Graeme O'Neill
They seek it here, they seek it there, but the Scarlet Pimpernel
molecule that spreads RNA-induced gene-silencing through plants, and confers
systemic protection against virus infections, remains elusive.
In a series of elegant experiments with grafted Arabidopsis seedlings,
CSIRO Plant Industry molecular geneticist Peter Waterhouse and Dr Bernie
Carroll of the Department of Biochemistry at the University of Queensland
have identified how the message is transmitted, and determined that it is
probably not a small RNA molecule.

The message may yet turn out to be a larger RNA molecule, but it
appears to work at the level of the gene, via epigenetic silencing rather
than the now-familiar machinery of RNA-induced silencing complexes (RISCs).

A decade ago, Waterhouse and CSIRO colleague Dr Ming-Bo Wang performed
a seminal experiment in tobacco which established that plants have a
cell-based defence against viral infections.

Carroll developed a system for micrografting Arabidopsis, and then
began a series of collaborative experiments with various rootstock-graft
combinations.

Their observations not only illuminate the 'spreading silence'
phenomenon, but may have explained how the practice of tissue-culturing
meristem tissues from perennial plants like grapevines and citrus can
eliminate persistent viral infections, restoring them to full genetic health
and productivity.

Waterhouse suspects some perennial plants have exploited a quirk of
RNAi, exclusive to plants, to achieve extraordinary longevity. These
near-immortal plants have survived for thousands, even tens of thousands of
years, untroubled by virus infections or epigenetic reprogramming that could
disrupt their fitness.

Viral protection

RNAi continues to transform the biological sciences - it is
undoubtedly the most powerful new tool for exploring and manipulating plant
genomes in three decades.

But it seems there is nothing new under the sun - researchers first
observed and harnessed its potential to protect crop plants against viral
infections more than 75 years ago.

In 1929, pioneering plant pathologist Harold McKinney, working on a US
Department of Agriculture farm that later became the site of the Pentagon,
began exploring the enigmatic phenomenon of cross-protection.

McKinney infected tobacco plants with a mild strain of tobacco mosaic
virus (TMV), and found that, by some act of green magic, they became fully
resistant to pathogenic TMV strains. Agronomists took up the technique,
using it to protect crops like tobacco, citrus, cucurbits, grapevines and
pawpaws against viral infections.

Local inoculation with a virus somehow protected the entire plant.
Many hypotheses were advanced, but the mechanism remained an enigma until
Waterhouse and Wang's seminal experiment in tobacco in Canberra in 1997.

By 1993, the CSIRO researchers were convinced that the release of the
double-stranded RNA (dsRNA) genome of an invading virus triggers a
cell-based defensive mechanism that disrupts the virus' replication.

In 1986, US plant virologist Dr Roger Beachy had made tobacco plants
resistant to tobacco mosaic virus (TMV) with a transgene coding for the TMV
capsid protein. The prevailing idea was that over-expressing coat proteins
blocked infection by disrupting the assembly of new virions.

Waterhouse and Wang experimented with Beachy's technique, but like
many others, obtained inconsistent results.

Sometimes, transgenic plants exhibited resistance even though they
expressed little or no coat protein from the target virus. A vital clue
emerged in 1992, when US plant virologists Bill Dougherty and John Lindo
showed that the viral RNA was being degraded before it could be translated
into protein.

The anti-viral defence was triggered, not by viral proteins, but by
the virus's double-stranded RNA genome.

In 1997, Waterhouse and Wang developed two transgenic tobacco lines,
each containing a transgene coding for one strand of an RNA sequence from
Potato Virus Y (PVY). The parents were susceptible to TRV, but when they
were crossed, 25 per cent of their F1 progeny were fully resistant.

The resistant plants had inherited both transgenes. The messenger RNAs
had base-paired, forming a double-stranded RNA (dsRNA) molecule. Somehow,
the dsRNA molecule directed the degradation of the corresponding sequence in
the virus itself, when the plants were inoculated. Anti-viral RNAi
transgenes are typically expressed constitutively ('always on') in all the
plant's tissues - yet in plants and animals, injecting short interfering RNA
(siRNAs) induces a similar protective effect that spreads throughout the
plant's cells.

In 1998, Waterhouse and Wang provided one of first detailed
descriptions of the machinery of RNA interference in plants.

Protein-RNA assemblages called RNA-induced silencing complexes
(RISCs), mediate RNAi by cleaving double-stranded RNA molecules into
fragments that vary from 21 to 24 nucleotides in length.

RISCs retain the fragments, which serve as templates for identifying
complementary RNA sequences from viruses, or mRNAs from genes.

In animals, the RNA-cleaving endonucleases are encoded by one, or at
most two, Dicer genes.

Waterhouse and Wang showed that dicotyletods like Arabidopsis have a
basic complement of four specialised Dicer-like genes; poplar has five.
Monocots typically have five; rice has six.

Waterhouse says the length of each fragment is a clue to which Dcl
endonuclease produced it, and its role. Dcl1 yields 21nt microRNAs that
regulate development. Dcl2 produces 22nt RNAs for antiviral defence.

Dcl3 makes 24nt RNAs that direct the methylation and histone
deacetylase reactions that regulate gene activity by remodeling chromatin.
And Dcl4, like Dcl1, makes 21nt RNAs that play the major role in virus
defence. It is the Dcl gene that processes synthetic, hairpin RNAs to prime
plant cells to resist viruses.

Waterhouse's team located Arabidopsis mutants for each gene, then
crossed them to produce double mutants with all possible permutations of
Dcl2, Dcl3 and Dcl4, including a triple-mutant knockout. Multiple
developmental abnormalities left Dicer-like1 knockouts sickly and infertile.

Waterhouse and Carroll then performed grafting experiments with
various stock-scion combinations to identify the molecule that spreads the
gene-silencing effect from tissue to tissue, and to determine its mode of
transmission.

They created rootstocks by inserting an RNAi 'hairpin' gene into each
combination mutant line, targeting expression of green fluorescent protein
(GFP), the standard marker of gene activity in plants and animals. They also
inserted a GFP transgene into the rootstock plants, but the anti-GFP
transgene blocked its expression, producing a normal phenotype.

The scions were taken from transgenic plants expressing GFP, whose
tissues glow green under blue light - normal, green tissues lacking GFP
appear red under blue light.

As rootstocks, they used the four combination mutants, including the
triple mutant, after engineering each with a hairpin RNAi transgene designed
to knock down expression of GFP in the scion.

Mystery messenger

Before experimenting with the transgenic stock-scion combinations,
they performed a 'dry run' to determine how long it took the grafted plants
to re-establish fluid flow through the graft.

By injecting a fluorescent green dye into the rootstocks of normal
grafted plants, they established that it took five days for severed phloem
tubes to reconnect, allowing the dye to flow into the graft.

Assuming the mystery RNAi 'messenger' was transported in the same
manner, the earliest any GFP-silencing effect should be detected was five
days post-grafting.

They left the scions on the rootstocks for varying lengths of time
after phloem tube reconnection occurred at five days, then beheaded the
grafted plants and implanted the scions on nutrient media.

They also made time-lapse videos of the action. "If you grow the
plants without roots, you still see GFP silencing, showing that the signal
continues to propagate through the tissues, even when it is disconnected
from its source in the rootstock," Waterhouse says. "So the signal doesn't
need to be constantly provided - it self-perpetuates after the initial
pulse."

The experiment confirmed that the signal travels via the vasculature,
not the plasmodesmata - microscopic channels in cell membranes, through
which adjacent cells exchange ions and small molecules.

"This was reminiscent of work by Herve Vaucheret in 1997," Waterhouse
says. "He showed that if you took the top part of an unsilenced tobacco
plant, and put it on top of a silenced plant, the top part would be
silenced.

"So it was predicted that the signal consisted of small RNAs moving
through the vasculature, although it was not known at the time that there
were four Dicer-like genes, producing different-sized small RNAs. The
mutants allowed us to ask the right questions."

It had been suggested that the 24nt RNA from Dcl3 was the messenger.
The Dcl3 mutant rootstock produces 21 and 22nt RNAs, but not 24nt RNAs - yet
GFP silencing still occurred.

Rather than test the other double-mutants, the CSIRO researchers used
the triple mutant, which expresses only Dcl1. To their enormous surprise, it
still switched off GFP in the scion.

The experiment didn't rule out the possibility that Dcl1's 21nt
molecule was the message-bearer, but that seemed unlikely, because the
tissues of the triple-mutant rootstocks continue to fluoresce bright green,
showing that no small RNAs - including Dcl1 - were being made against GFP

Yet the triple mutant rootstocks were still sending a GFP-silencing
signal that switched off fluorescence in the scion. If the signal wasn't a
small RNA, what could it be?

"Our working model is that it's not a small RNA, but some longer RNA
made from the hairpin. Whether it's the entire hairpin is unclear.

"And that's the current state of play: we know a hairpin creates it,
and it's almost certainly not a 'Diced' product." "Spreading silence" is not
unique to plants - it was PhD student Su Guo's observation of the phenomenon
in the nematode C. elegans in 1994 that alerted US geneticists Professor
Craig Mello and Professor Andy Fire to the presence of a systemic, RNA-based
gene-silencing phenomenon in nematodes, earning them the 2006 Nobel Prize
for Medicine and Physiology.

When used as a rootstock, the Dcl3 mutant failed to transmit a
GFP-silencing signal to a GFP-expressing scion. Yet, when used as a scion,
it continued to glow green. So a Dcl3-diced RNA could not be the messenger.

"It was unable to receive the signal and convert it into GFP
silencing," Waterhouse says. "That was a real eye-opener."

If the elusive signalling molecule wasn't a small, diced RNA, some
other mechanism had to be at work: possibly an epigenetic effect, operating
at the level of the GFP gene, or its messenger RNA, that repressed
transcription.

Five days after dye was injected into the rootstock, it began moving
into the aerial parts of the plant, including the leaves.

By this stage, the scion had developed five or six leaves, from tiny
bumps on the stem, called leaf primordia, that were already present at the
time of grafting. These unsilenced leaves expressed GFP.

But the seventh and all subsequent primordia, newly differentiated
from meristem tissue, produced red leaves: the GFP-silencing signal had
reached these tissues.

And while the evidence was that it had arrived via the vasculature,
the phloem tubes ended in disorganised tissue below the meristem, as the
cells in the elongating shoot continued to differentiate.

It was as if the genes detecting the signal were silencing GFP
expression by some epigenetic mechanism blocking the transcription
machinery's access to the gene's promoter.

But this process clearly did not occur in pre-formed leaves which
still glowed green - they were already committed to express GFP.

So silencing must be occurring in the undifferentiated meristem cells.

The CSIRO team's working model involves three layers of meristem
cells. The third, lowermost layer forms the elongating vasculature as the
shoot grows, while gene-silencing occurs in the second "action" layer.

As the cells of the second layer divide and differentiate, they are
pushed sideways and upwards, carrying the third layer of undifferentiated
meristem cells on top of the growing shoot.

"If the signal is coming up the vasculature and flooding the region in
which the stem cells are located, they are possibly perceiving the signal
and silencing the GFP gene, so the tissues no longer glow green," Waterhouse
says.

"Now imagine that these stem cells are dividing and producing the
tissues for the next. So what we might be seeing, instead of the mobile
signal spreading through the leaves, is that it is converting the stem cells
and the differentiated tissues derived from them to the same state, so the
leaf is red instead of fluorescent green.

"We seem to have stumbled on some epigenetic process going on in
meristem cells.

"Interestingly, if you do a graft, then cut off the head and grow it
in nutrient media, it grows poorly, producing apical growth that throws out
leaves all over the place, but no flowers. But if we make the graft lower
down, the top part of the graft throws out lateral green roots, that
continue to grow, allowing the plant to grow and flower."

With this approach, Waterhouse's team made a plant that produces a
pulse of the silencing signal, then allowed it to grow normally and set
seed. Arabidopsis seed forms in small pod-like structures called siliques.

When they broke open the siliques, the inner surface was still
silenced, i.e. red - GFP silencing had occurred. But the newly formed seed
fluoresced bright green. The silencing signal had been lost, or erased, from
the germline cells that formed the seed.

"The more boring possibility is that the signal is lost during
meiosis, when the pollen or the ovule forms. But curiously, if you look at
the forming floral parts in the silenced plants, you can see that GFP is
actually 'on' in the younger flowers - and it seems to be 'on' just as
strongly in the pollen cells and ovules.

"So it doesn't seem that the silencing signal is erased during
meiosis."

Insurance policy

The more intriguing possibility, says Waterhouse, is that the plant
maintains a separate population of undifferentiated, virginal meristem
cells. (He was delighted to find that French researchers had first observed
this effect half a century ago and with typical Gallic panache, dubbed it
meristem detente. Needless to say, the idea was pooh-poohed at the time.)

These cells divide and give rise to floral meristem cells, which in
turn form the floral organs. But the virginal meristem cells remain
unchanged and insulated against malign influences within the sanctum
sanctorum of the floral meristem.

If so, these cells are the plant equivalent of the immunologically
privileged germline cells that give rise to sperm and ova in animals - they
are a form of insurance for the next generation against viral infections,
and potentially deleterious epigenetic influences.

They are now designing experiments to test the hypothesis, which they
believe makes evolutionary sense.

"Tissue culture uses these meristem cells - it's a way of clearing
virus infections from clonally propagated plants," Waterhouse says. "It's
possible that some of these virginal cells have been maintained unchanged
for millions of years."

But it is now clear, the CSIRO researchers say, that whatever spreads
the gene-silencing or virus-quelling signal through plant tissues involves
more than simple diced RNA molecules. The immortals

Humans in industrialised nations typically live around 80 years before
succumbing to cardiovascular disease, cancer or other diseases of ageing.

The oldest human on record, Frenchwoman Jeanne Calment, died of heart
failure in 1997, aged 122. More commonly, cancer is the ultimate price of
human longevity: new research supports a hypothesis that many tumours and
leukaemias arise from mutations in "immortal", pluripotent stem cells that
constantly renew the body's tissues and organs.

Even by the measure of Jeanne Calment's extreme longevity, some woody
plants, both conifers and flowering plants, are virtually immortal. They can
continue to regenerate from meristem cells - the plant equivalent of stem
cells - for millennia, without accumulating genetic errors, or plant
tumours. How?

In 1937 Tasmanian bushman Denny King discovered a new species of
lomatia, Lomatia tasmanica, (now popularly known as King's holly), a distant
cousin of the waratah, in the island's perennially wet south-west.

The shrubby plants form a linear population that wends its way through
more than 1.6 kilometres of dense temperate rainforest.

Cytogenetic analysis revealed they are genetically identical: they are
all clones of a rare, triploid progenitor whose dull pinkish red flowers -
unique in the trans-Pacific genus - are sterile and set no seed.

Morphologically identical, sub-fossilised leaves found in late
Pleistocene sediments 8.2km away have been radiocarbon-dated at 43,000
years - not coincidentally, the practical limit of radiocarbon dating, so
they could even be older. So King's Holly is at least 10 times older than
"Methuselah", a 4773-year old bristlecone pine (Pinus longaeva) in
California's White Mountains.

Methusaleh's status as the world's oldest tree is contestable. On the
exposed, rain-swept upper slopes of Mt Read, near Rosebery in western
Tasmania, grows a one-hectare stand of gnarled, stunted Huon Pines
(Lagarostrobus franklinii).

Some larger trees in a sheltered grove are more than 2000 years old.
All trees are genetically identical, and male - a clone.

No other Huon grows within 20km of Mt Read. Pollen from the sediments
of a glacial tarn, Lake Johnston, downslope of the krumholtz ('twisted
wood') pine, is of the same genotype. It has been radiocarbon-dated at
10,500 years.

But even Mt Read's venerable Huon pine may be younger than the
diminutive Mongarlowe mallee, Eucalyptus recurva, discovered by an amateur
botanist near Braidwood, in south-eastern NSW.

There are only four tiny, isolated populations, all apparently clones
of long-vanished original trees that survived the last glacial period. The
small leaves are the thickest of any eucalypt, and are studded with large
oil glands - adaptations to killing frosts.

Unlike Kings Holly and the Mt Read Huon pine, the Mongarlowe Mallee
sets seed, but produces no new seedlings. They germinate only after being
refrigerated for months in the laboratory, at freezing temperatures. Since
the last glacial period 12,900 years ago, winters may not have been cold
enough - nor long enough - to promote germination.

Peter Waterhouse's research into the mysterious mechanism of spreading
RNA interference hints at the existence of an RNA-mediated mechanism that
insulates meristem cells in the growth shoots or epicormic buds of these
"immortals" against time and pathogenic tide, so they continue to replica
with extraordinary fidelity.
[www.biotechnews.com.au]