Thursday, March 28, 2024
General Agriculture

Production of Disease and Pest Resistant Plants

Control of Plant Viruses: There are currently three approaches to controlling plant viruses. Pesticides are used to control the invertebrate vector. This can be environmentally damaging (see the white insecticide deposits on the rice) and many insects develop resistance to the insecticides.

The second is clean agronomic practices such as removal of sources of infection. This can be effective for perennial crops but in others such as attempts to control Cacao swollen shoot virus did not succeed.

This approach does not work for many annual crops in tropical countries where young and old crops are grown side-by-side. The third approach is the breeding of resistance into the crop; this can be either by conventional breeding or by GM.

In conventional breeding the resistance can be to either the virus or the vector. However, the great variability of many viruses results in the plant breeder having to play “catch up” with new virus variants that break the resistance gene.

This figure shows an example of this with the reaction of various genes bred into tomato against variants of Tomato mosaic virus (related to Tobacco mosaic virus).

In practice, when one resistance gene was widely deployed a new strain of the virus arose which broke the resistance. This has led to considerable efforts in applying GM approaches to controlling plant viruses.

In some of the earliest experiments with transgenic plants it was shown that the transgenic expression of some viral sequences could confer resistance to the cognate virus.

The initial experiments on transgenic virus resistance involved the expression of the viral coat protein (termed coat-protein protection); subsequently, expression of other viral gene products, either unmodified or mutated in important regions, have given some protection.

The results from some of these protein-expressing systems did not make sense as resistance was also obtained using some non-coding sequences (the controls on the experiments).

Thus it appeared that the expressed nucleic acid was playing an important role. In some of these experiments, the expressed RNA was complementary (antisense) to the viral mRNA, in others it was in the same sense.

This, and other observations, led to the realization that plants (and other organisms) have a previously unrecognized defense system against “foreign” nucleic acids – termed GENE SILENCING.

There are two inter-related forms of gene silencing, one that operates on the chromosomal DNA (TRANSCRIPTIONAL GENE SILENCING) and the other on the RNA in the cytoplasm (POST-TRANSCRIPTIONAL GENE SILENCING or PTGS).

PTGS, also known as RNA silencing, quelling (in fungi) and RNAi (RNA interference) (in animals), has proven to be a very important feature in transgene technology.

At some stage in the replication of the “foreign” RNA, double- stranded RNA (dsRNA) is formed and the plant (or animal) recognizes this as being foreign.

The right-hand part of the figure shows what happens to this dsRNA. It is targetted by an enzyme called DICER which cuts it into pieces of 21 to 25 nucleotides (termed small interfering RNAs or SiRNAs).

These then combine with various proteins to form to form a macromolecules termed the RNA-induced silencing complex (RISC). Proteins in the RISC unwind the double-stranded SiRNAs which then give single-standed SiRNAs of both polarities.

The SiRNA then anneals to the target RNA (which has the same sequence as that from which it originated) making further dsRNA which is in turn targetted by DICER. Thus the “foreign” single- stranded RNA is degraded.

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The importance of ds RNA in the RNA silencing process has led to constructs that transcribe to give this molecule. In this construct the coding sequence is duplicated to give an inverted repeat, the two parts being separated by a non-coding region (intron).

This approach is opening up a whole new transgenic strategy not only against viruses but also using RNA silencing to “switch off” the expression of undesirable genes, e.g. for allergenic gene products.

There are several examples of transgenic protection against viruses in the field. These two examples were intended to involve coat protein protection but in the light of finding on RNA silencing, there may be uncertainty as to how they operate.

However, as pointed out below, it is important in making a biosafety determination to know which mechanism is involved.

Several main areas of concern have been raised about the deployment of plants transgenic in virus sequences.

The main ones are: Interactions with other viruses; Suppression of gene silencing; Spread of transgenes to other plants; Possible effects on humans and other animals; Stability of the transgene.

Although the transgene viral sequence will give protection against the donor virus (and possibly closely-related viruses) it will not do so against unrelated superinfecting viruses.

Thus there is potential for at least three types of interaction between the transgene viral sequence and the superinfecting virus:

Firstly, as the coat protein of a virus can determine the specific interactions with the vector (insect or nematode) the concern is that the transgenically- expressed coat protein can encapsidate (termed heteroencapsidate) the genome of the superinfecting virus thus changing the transmission properties of that virus.

This happens occasionally with natural double infections of non- transgenic plants but there is no evidence that it is more prevalent with transgenic plants.

Secondly, some combinations of viruses cause more severe symptoms than the sum of those of the two original viruses – termed synergy. The concern is that the transgene will interact with the superinfecting virus exacerbating the symptoms.

Both these concerns relate to transgenes that produce proteins. Thus the use of the RNA silencing approach is likely to be much more efficient at protecting plants against virus infection and so these concerns should not be important in the future.

The third and main concern about the use of viral transgenes that produce just nucleic acid is that there could be recombination between the transgene RNA and that of the super infecting virus giving rise to a new “super virus”.

Production of Disease-Free and Disease/Pest-Resistant Plants

This would happen during replication of the viral nucleic acid, usually by the replicase enzyme switching strands. It would only potentially cause a problem if the recombinant virus was viable and had a selective advantage over the superinfecting virus.

There are several examples of recombination between two viruses in non-transgenic situations resulting in new viruses; one case is the devastating Uganda variant of African cassava mosaic virus that devastated the cassava crop in East Africa in the 1990s.

There are further concerns of potential hazards relating to plant viruses. The gene silencing (RNA silencing or RNA interference) phenomenon is raising two concerns about viruses and transgenic plants.

Firstly, as mentioned above, the CaMV 35S promoter is widely used and infection of oilseed rape with CaMV has been shown to silence the expression of herbicide resistance expressed from that promoter.

The second concern relates to a property of successful viruses. As RNA silencing is a defense mechanism against “foreign” nucleic acids successful viruses have to suppress it to be able to overcome this host defense.

Various viral genes have been identified as suppressors of RNA silencing. The question is does this suppression of gene silencing occur in an infection of a transgenic plant switch off a silencing construct?

Further concerns about transgenic protection against viruses are more general and include possibility of spread of the transgene to other plants causing effects to the environment (virus resistant plants in the ecosystem), possible effects of the expression of viral sequences on humans and other animals (e.g. allergies) and stability of such transgenes.

As much is known about the detailed molecular biology it should be possible to develop systems to minimize or obviate any hazard.

Thus, one can make viral constructs that do not contain a potentially hazardous sequence, for instance the amino acid sequences that control insect vector specificity or nucleic acid sequences that are involved in recombination. This is termed “sanitizing the construct”.

However, most of these hazards are hypothetical and the best approach is to monitor releases, a subject that will also be discussed later.

1. Fungal Pathogens

There is a wide range of fungal diseases that can affect all parts of plants and. Some fungi cause leaf damage either leading to a quick death of the infected leaf as in potato blight or slower death but significant effects on the plant as in mildews and rusts.

Fungi such as Botrytis cause rots in storage organs and fruits. Scab fungus damages potatoes with loss of quality and storage ability; similarly, mildews can cause loss of quality in fruits such as grapes.

Cereals can be affected by smuts and ergot; ergot is very important as it contains various alkaloids, ingestion of which can cause constriction of blood vessels, abortions, hallucinations and mental aberrations.

Fungi can also affect and kill trees. One of the most notorious fungi is Potato late blight, which caused the Irish Potato famine leading to a mass migration primarily to the USA. Fungal infections can also produce mycotoxins.

Conventional Control of Fungal Pathogens

There are currently three approaches to controlling fungal diseases. The most widespread is the application of fungicides of which there are three types: sterilants and fumigants which are usually used to sterilize soil or fumigate enclosed spaces; protectants which are sprayed to prevent infections; therepeutics which are used to “cure” infections.

There has been a continuous increase in the use of fungicides over the last 20 years or more (in the USA rising from 19.5 million kg in 1983 to 35.5 million kg in 1993) and the use of this input is increasing worldwide.

Read Also : Introduction to Plant Pests and Insects

There are several problems arising from the continuous use of fungicides: resistant variants of pathogenic fungi are selected, there is an unquantified ecological impact; there are food safety concerns and there are potential health hazards for the farmer.

The term “pest” was originally applied to animals (insects, nematodes, mammals and birds) that caused crop losses; the term “disease” applied to viruses, bacteria and fungi. However, the term “pesticide” is now generally used for chemicals that cause biotic losses in crops.

The second approach to controlling fungal diseases, as with virus diseases, is good agronomic practice such as removing sources of infection or growing the crop either in places where the pathogen does not occur or at a time of the year when it is not spreading. The details of this approach vary with pathogen, agronomic system, climate and other conditions.

Thirdly, conventional breeding for resistance to specific fungal diseases has been used for many years. There are three major genetic traits that are used in breeding programmes:

Resistance can be given by various characters of plant structure such as a thick and waxy cuticle (outer skin of the plant leaves and stem) or a leaf structure that does not accumulate water drops (the spores of many fungi require water for germination).

When a fungus infects a plant there is chemical communication between the two, the fungus determining a suitable host and the plant trying to resist fungal infection. Some resistance genes enhance the plant defence.

Although not much is known about most of such plant defences one has been studied in detail. This is the hypersensitive response in which the plant responds by killing off its cells around the initial stages of infection.

This response is due to the interaction of a plant resistance R gene and the fungal elicitor gene. The understanding of this interaction has led to the gene-for- gene theory, one fungal (elicitor) gene product interacting with one host (resistance) gene product.

Many plant R genes (which can also give resistance to bacteria and viruses) have been characterized and shown to have various basic features in common such as nucleic acid binding domain (NB) and leucine-rich repeat (LRR).

One of the problems with the gene-for-gene situation is that that a small change in the fungal elicitor gene can overcome the plant R gene and thus resistance breaks down.

The breeder is having to find new sources of R genes for variants of fungal pathogens – the actual deployment of an R gene into the field can often select resistance-breaking variants.

The problem then comes of finding sources of R genes in species that can be successfully crossed with the crop of interest.

Genetically Modified (GM) Control of Fungal Pathogens

As with viruses, an understanding of the interactions between the pathogen and host has led to developing transgenic approaches to protecting crop plants against fungal infections.

However, the interactions are more complex than those of viruses and to date (2006) there have been no commercial releases of transgenic fungus-resistant plants. There are several approaches being used in laboratory studies on conferring protection against fungi with transgenes.

R genes can be isolated from resistant varieties of the same species or from closely related species and put into the desired crop.

An example of this is the resistance gene to potato late blight isolated from a related species and inserted into potato. The colinearity or synteny of genes in chromosomes of related species makes the identification and isolation of such genes easier.

This approach of the transgenic movement of R genes between plants has several advantages such as shortening breeding programmes, bypassing sexual incompatibility barriers and being able to “stack” R genes (i.e introduce several R genes into an elite variety).

Because R genes have a common structure and their specificity is due to minor variation in that structure, another source of resistance genes is to modify the R gene in the laboratory.

The interactions between the fungal elicitor and the R gene lead to a cascade of metabolic reactions resulting in hypersensitivity and other host responses.

Modification of this pathway is showing some potential for conferring resistance. Similarly, the enhancement of other plant responses to pathogen infection is showing promise.

Many plants produce small peptides that have anti-microbial properties. Several of these have been isolated, characterized, their genes identified and inserted into the crop species.

Similarly, anti-microbial peptide genes from dahlia and onion have been put into bananas giving protection against the devastating black Sigatoka fungus.

Various other genes are being tested for protecting transgenic plants against fungal infection. The cell walls of fungi are made of chitin and glucans and one of the most promising approaches is to attack the fungal cell wall with chitinases and gluconases.

2. Bacterial Pathogens

Bacterial pathogens are not very important in temperate countries but can be very important in tropical countries. Plant pathogenic bacteria cause a variety of symptoms usually wilts and rots; one, Agrobacterium, causes crown gall due to transfer of part of a plasmid DNA to the host.

The current control measures are similar to those of fungal pathogens comprising agronomic approaches such as using clean planting material and planting at times or places to avoid the disease together with breeding for resistance.

Several R genes are proving important in breeding programmes. Some studies are being made on contolling bacterial pathogens by Genetic modification of crop plants using approaches similar to those described above for fungal pathogens.

One recent success has been the transfer of the rice bacterial blight R gene, Xa21, from a basic rice variety into elite cultivars where it gave broad spectrum resistance to strains of the pathogen. Some Xa21 transgenic lines should be released in China during 2006 or 2007.

Biosafety Considerations

The main biosafety concerns about crop plants transgenically protected against fungi and bacteria are on the food and health safety of novel gene products (e.g. chitinases) and spread of resistance genes into the environment.

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Agric4Profits

Benadine Nonye is an agricultural consultant and a writer with over 12 years of professional experience in the agriculture industry. - National Diploma in Agricultural Technology - Bachelor's Degree in Agricultural Science - Master's Degree in Science Education... Visit My Websites On: 1. Agric4Profits.com - Your Comprehensive Practical Agricultural Knowledge and Farmer’s Guide Website! 2. WealthinWastes.com - For Effective Environmental Management through Proper Waste Management and Recycling Practices! Join Me On: Twitter: @benadinenonye - Instagram: benadinenonye - LinkedIn: benadinenonye - YouTube: Agric4Profits TV and WealthInWastes TV - Pinterest: BenadineNonye4u - Facebook: BenadineNonye

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