Scientists at the John Innes Centre (JIC) Norwich [1], report a new technique for identifying and isolating genes that they estimate reduces the time and cost involved to one tenth, or less, of existing techniques. The new technique (transcript-based cloning[2]) relies on scientists taking ‘snapshots’ of gene activity in plants. By comparing snapshots, taken from normal plants and plants with genetic mutations, they are able to identify and isolate the gene affected by the mutation. The technique has enormous potential to accelerate and ease the identification and isolation of important genes, not only in plants but in other organisms. The scientists have demonstrated the power of the technique by using it to identify DMI3[3], a gene that is part of a plant communication and control system that recognises, and responds to, the presence of bacteria and fungi that are able to form symbiotic relationships with plants. Thus the report also has important implications for scientists’ efforts to understand how plants and micro-organisms converse in order to cooperate.

“We believe the technique we have developed is a major breakthrough” said Dr Giles Oldroyd (leader of the research team at JIC), “we have done in four months what would have taken 4 years using other methods, and at a tenth of the cost. The way the technique works means that it is not only a relatively quick and cheap way to identify and isolate genes, but it also makes it practical for us to go looking for genes in some important, but genetically complex, crop plants where using conventional gene detection methods would, if not actually technically impractical, be a hugely laborious and costly process”.

The JIC team worked in collaboration with a group in the Department of Biological Sciences at Stanford University, US, on Medicago (Medicago truncatula) a member of the Legume family and a relative of alfalfa, peas and beans. The legumes are of particular interest because they are able to form a specialised mutually beneficial partnership (symbiosis) with bacteria (called Rhizobia) and this enables them to make their own nitrogen fertiliser (nitrogen fixation) [4]. In developing the technique the scientists compared normal Medicago plants with Medicago plants that they knew were unable to establish a symbiotic relationship with Rhizobia, because of a mutation in a gene called DMI3. DMI3 is involved in another intimate and beneficial relationship between plant’s and micro-organisms, this time with soil-living fungi in so-called arbuscular mycorrhizal associations[5]. Plants defective in the DMI3 gene are unable to form mycorrhizal associations. A better understanding of these two symbiotic interactions should allow scientists to develop crops less dependent on fertiliser application.

Transcript-based cloning is an innovation based on DNA microarrays and the predicted instability of RNA transcripts from genes damaged by mutation. A DNA microarray is a small glass slide covered with an ordered pattern of microscopic dots. Each dot consists of multiple copies of fragments of an individual gene. The team prepared a microarray that was made up of nearly 10,000 dots, each dot representing a different gene known to be active in the roots of normal Medicago plants.

A microarray is used to detect the primary product of active genes (RNA transcripts), and thus indicates which genes represented on the microarray, are active in the plant i.e. which genes in the plant are making RNA transcripts. The JIC scientists predicted that genes damaged by mutation would make RNA transcripts that themselves were damaged and that these transcripts would be unstable. They expected that when they tested extracts from the mutant plants on the microarray, they would be able to detect the reduced amounts of RNA transcripts from the damaged gene. When they compared the pattern of gene activity in the normal and the mutant plants they found three genes that did not seem to be making stable RNA transcripts in the mutant plants - one of these genes subsequently proved to be DMI3.

“We are very excited about the potential of transcript-based cloning”, said Dr Oldroyd. “It is a relatively quick and cheap technique that is applicable to gene isolation in many different systems, from humans to bacteria, but it is especially significant because it potentially makes gene identification in complex genetic systems much easier than at present”.

The report is published in the international scientific journal, Proceedings of the National Academy of Sciences and online in the PNAS Early Edition.

Intellectual Property associated with this discovery is assigned to Plant Bioscience Ltd. [6].

BACKGROUND

[1] The John Innes Centre is an independent, world-leading research centre in plant and microbial sciences. The JIC has over 850 staff and students. JIC carries out high quality fundamental, strategic and applied research to understand how plants and microbes work at the molecular, cellular and genetic levels. The JIC also trains scientists and students, collaborates with many other research laboratories and communicates its science to end-users and the general public. The JIC is grant-aided by the Biotechnology and Biological Sciences Research Council.

[2] Transcript-based cloning is an innovation based on DNA microarrays and the predicted instability of RNA transcripts from genes damaged by mutation. A microarray is used to detect the primary product of active genes (RNA transcripts). A DNA microarray consists of an ordered pattern of microscopic dots on a glass slide. Each dot consists of multiple copies of fragments of an individual gene.

The team used a microarray representing 9,935 genes known to be expressed in roots of normal Medicago plants. (This represents about one third of the total number of Medicago genes). Labelled RNA transcripts were prepared from normal and DMI3 mutant Medicago plants and the patterns of transcript binding to the microarray compared. As predicted, RNA transcripts from the mutant gene were unstable and therefore had a low abundance in the extract from the mutant plants. These low levels of transcript could be detected on the microarray and the relevant genes cloned for examination.

[3] DMI3 was identified in Medicago truncatula. DMI3 encodes a calcium/calmodulin-dependent protein kinase. Similar mutations are found in pea (sym9). The DMI3 class of proteins are well conserved among plants that form associations with nitrogen-fixing rhizobia and mycorrhizal fungi.

DMI3 mutants respond to bacterial Nod factors (nodulation signals) with calcium spiking and root hair swelling (early changes in the root associated with nodulation) but do not show changes in gene expression levels or cell division needed for nodule formation.

The DMI3 gene makes a protein which is not involved in the plant’s recognition or early response of the bacteria, but is an important part of the communication system that relays the initial response through the plant cell. Medicago plants with a damaged DMI3 gene can respond to the presence of bacteria and show some of the early responses associated with making root nodules. However, the process stops prematurely and the relationship fails.

DMI3 is also involved in another intimate and beneficial relationship between plant’s and micro-organisms, this time with particular soil-living fungi in so-called arbuscular mycorrhizal associations.

[4] The nitrogen-fixing symbiosis (a relationship in which both partners benefit) between legumes and Rhizobium bacteria is thought to be a relatively recent evolutionary development. The root nodules (that accommodate the bacteria) can be thought of as the natural bio-reactors in which the conditions for nitrogen-fixation are maintained.

This unusual and highly specialized symbiosis enables the bacteria to take nitrogen gas from the atmosphere and convert it into nitrate and ammonia (nitrogen fixation), which are absorbed and used by the plant. The plants are effectively able to make their own fertilizer as a result of this partnership. In return the bacteria are able to absorb and use sugars produced by the plant.

The relationship begins with plant roots producing a chemical signal that stimulates any Rhizobia bacteria, in the surrounding soil, to produce their own signals. When the plant detects the bacterial signal a cascade of changes occur in the plant root. This eventually results in the bacteria invading the roots and taking up residence in specialized structures, called root nodules, which are produced on the plant’s roots. The bacteria in the root nodules then begin to fix nitrogen.

Plants cannot use nitrogen directly from the atmosphere, but rely on it being converted to a usable form, such as ammonium, in order to use it. The symbiotic relationships between legumes and rhizobia account for 65% of the global nitrogen that can be used by other plants (when the legume dies) or animals (when the legume is eaten). The manufacture of ammonium fertilizer is an energy intensive process while the application of manufactured, and natural, nitrogen fertilizers can have adverse environmental impacts (eg. run-off to ground water). The ability of legumes to fix atmospheric nitrogen is the reason they are included in farm rotations - as a means to return nitrogen to the soil.

[5] Arbuscular mycorrhiza are associations between roots and specific soil-living fungi, which are commonly found among many higher plants, including major crops. The associations are of interest because they increase the plant’s ability to take up nutrients from the soil. Typically, the hyphae of a mycorrhizal fungus that come into contact with the root surface of a compatible plant, will penetrate the root epidermal cells and enter the root cortical cells. Here a specialised structure within the cell develops that accommodates the invading hypha of the fungal partner and a stable long-term symbiosis is established. This is an ancient symbiosis having been found in fossils of early land plants.