RNAi and the sound of silence

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 (see page 4).

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.

---PB---

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."

---PB---

"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.

---PB---

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.