Plant diseases and plant defenses:

In today's class we will go over various plant defense mechanisms and microbial retorts to plant defenses. These notes are most certainly not inclusive but will provide you with an introduction to the fascinating topic of plant defenses....

I. How do fungi and other microbes hurt plants?

1. Chlorosis; wilting as a result of ammonia accumulation in tissues
Normally NO2==> glutamine but with enzyme inhibition instead becomes NH3--> which is toxic to the plant

2. Bacteria and fungi produce high levels of "plant hormones"

fungi--> gibberellins - known examples include the fungus which causes rice to grow at a hyper or 'crazy' rate - the study of which lead to the identification of gibberellin.

Various bacteria produce --> cytokinins.. elevated growth rates, metabolism.

This 'trickery' however is not exclusive to microbes; larvae of various insects also give off hormonal analogues which cause the plant to produce new vascular tissue in the area of the invader and which allows it to be fed as it develops. At the same time, plants do as we learned earlier, produce insect/animal hormones which cause them to develop at inappropriate times, so all is fair in ....

3. Wilting

Invaders produce polysaccharide gums which plug up xylem tissue and messes up stomatal control via hormones--> ABA. We discussed earlier root rot or damping off in seedlings when overwatering young plants. This encourages fungal growth and subsequent gumming off. As the plant wilts, we tend to water more, thinking it its lack is the cause of the wilt, and further encouraging fungal growth.

4. Metal and other ion chelators

Metal chelators sSteal' metal atoms and tie them up. Specific microbial compounds scavenge Fe and Cu in the plant cell - others can complex with Ca ( oxalic acid) causing grief with ion flow, metabolic reactions..

5. Break down cells walls with enzymes

degradative enzymes cause plant '' rots" via cell wall separation; includes the secretion of cellulase and pectic enzymes. Fungi are notorious for producing various pectinases which dissolve the glue that holds walls together.

6. Interfere with plant metabolism..examples given below

7. Interfere with membrane structure, making them leaky... mesophyll cells then leak out nutrients; control may center about ion flow ( Ca++ ) or with the structure of the membrane itself, especially the fatty component. Various compounds are known to affect lipid structure, and rearrangement will cause breakdown of membrane structure and regulation of flow through it.

 

II. How do plants defend themselves these detested invaders?

Defense mechanism are broken down into 2 major groupings: mechanisms which exist before infection and those which are activated with infection...

A. Those defenses which exist before infection are generally nonspecific. They include the prohibitins which help reduce microorganism development or inhibitins which increase in level after infection or become fully toxic with infection but in both cases exist in some form before infection

Pre infected or preformed defenses are generalized... they have not evolved in relation to the expression of a specific pathogen's product designed to harm the host.

How can a plant generate such a 'good for all contingencies' type of defense, given the pathogens can vary in wall structure ( fungi have cell walls made up of chitin vs. bacterial walls), biochemistry, water requirements and so on?

Generalized defense strategies:

1. Surface: As with animals, the skin or in this case the surface is a good first defense. Layering the epidermis with layers of wax prevents water-soluble products ( enzymes) from entering the plant. Obviously this system can't work where there are openings either natural ( stomata, root hairs, where mycorrhizae penetrate the plant root system) or where the surface tissue has been damaged.

Another structural defense are trichomes ( those surface hairs we saw earlier) , especially those who secrete various chemicals or enzymes.

2. Biochemical defenses

a. Enzyme or enzyme inhibitors: enzymes such as lysozymes or chitnases ( digest the chitin in the cell walls of fungal attackers) can help digest invaders. Proteinase inhibitors, lectins, thionins and polysaccharides which agglutinate or gel up the digestive enzymes produced by the invaders curb their action on the plant.

b. Preformed inhibitors used against other plants ( allopathic chemicals) can also act on microbes. These include phenols such as caffeic acid, caynogenic glycosides, and saponins.

Saponins include glycosylated sterols, tirterpenes, and alkaloids which are activated by the removal of a glucose unit. Once activated they complex with membrane sterols generating membrane pores in the invader. Of course some invaders have developed a mechanism to detox these saponins

Cyanogenic glycoside--> hydrogen cyanide which is an inhibitor of electron transport in respiration

subsequent breakdown generates highly reactive species: isothiocyanates, thiocyanates, nitriles

Cabbage plant--the stuff that gives it the smell of cabbage is isothiocyanate which is also the agent responsible for its' resistance. It is more resistant in higher concentrations. We've bred it out cabbage for a milder flavor and therefore the cabbage is now less resistant

Onions--Enzymes attack and become the compound that makes onions smell and eyes tear

B. Post infection defenses and elicitors:

1. Phytoalexins--low molecular weight, antimicrobial compounds that are both synthesized by and accumulated in plant cells after exposure to microorganisms

The existence of phytoalexins was postulated as early as 1905 by Ward's Toxin theory: plants produce toxins or antibodies to defend themselves... however proving the existence of the compounds involved was a difficult task.

Miller and Borga in 1941 proposed the Phytoalexin Theory--Plants produce chemical substances de novo at time of infection. They studied potato resistant to Phytophora infestations one of the most notorious fungal pathogens which changed the history of Ireland...

However not until 20 years later did Cruickshank + Perrin isolate a specific compound that exists in in peas known as pisatin

Generalizations:

  • compound inhibits development after infection
  • occurs only in living cells
  • compounds are nonspecific
  • resistance depends on how fast they can produce it.. in some cases precursors may be released from conjugates which speeds up defenses
  • lipid like or phenolic-->protein denaturation in fungi; bacteria
  • are now known to include a variety of compounds: sesquiterpenoids, phenylpropanoids (stilbenes, anthocyanins, isoflavonoids

2. Hypersensitive reaction

plants cells die immediately with contact with the fungal cells. Fungus needs live cells to live off--can't grow in dead cells--so fungi tries to extend out--but as soon as it does those cells die. Phytoalexins may help in signaling or causing cell death.

3. Rapidly forming wound cork

around injury caused by fungus cells adjacent to invasion site cells divide--differentiate cork saturated with suberin (wax) and lignins which are highly resistant to enzyme attack

 

4. Recent work suggests a series of reactions occur with infection:

Scheel, H. Hirt1, T. Kroj, W. Ligtering1, D. Nennstiel, T. Nürnberger, M. Tschöpe, W. Wirtz, and H. Zinecker

Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle (Saale), GERMANY and 1Institut für Mikrobiologie und Genetik, Dr.-Bohr-Gasse 9, A-1030 Wien, AUSTRIA

Plants apparently recognize potentially pathogenic organisms through receptors on their surface that specifically bind pathogen or plant-derived signal molecules, so-called elicitors, and thereby initiate signaling cascades activating a multicomponent defense response. Cultured parsley cells and various proteinaceous elicitors are the experimental system used to investigate the molecular mechanisms underlying recognition and signaling events in these processes.

The oligopeptide, Pep-13, from hyphal cell walls of the plant pathogenic oomycete, Phytophthora sojae, stimulates a multicomponent defense response in cultured parsley cells that is very similar to the response of intact plants to infection with this fungus. This elicitor is therefore believed to act as a recognition signal in this non-host plant/pathogen interaction, which is perceived by the plant cell through a plasma membrane receptor (1, 2). We are currently purifying the detergent-solubilized receptor by affinity chromatography. Furthermore, a second peptide elicitor of different structure has been detected in culture filtrate of Phytophthora infestans. This elicitor appears to initiate the same defense response as Pep- 13, however, through a distinct receptor site.

Early elements of the plant response are ( I've modified by adding some additional notes here emi)

  • ion fluxes across the plasma membrane (membrane changes & ion exchange (H+/K+ ATPase, Ca flux), This occurs in minutes...and may involve alkalinazation ( why? remember role of acidification of cell walls in growth?)

 

  • activation of a MAP kinase and
  • the production of reactive oxygen species (likely origin from NADPH oxidase complex in plasma membrane) and the oxidative burst *

Reactive oxygen species (ROS), likely have multiple roles

1. possible signals turning on defense

2. highly reactive, membrane permeable, and toxic to microbes and plant cells (superoxide anion ( O2*- )

thought to generate more reactive OH*, HO2*, and lipid peroxides. How would reactive oxidative molecules affect the invader?

3. H2O2 involved in structural modifications of cell wall

 

followed by

  • defense gene activation and phytoalexin accumulation.


Activation of the elicitor-responsive ion channels, the most rapid reaction, has been found to be necessary and sufficient for all other reactions of the plant cells (3, 4). Inhibition of elicitor-stimulated production of reactive oxygen species with diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase, blocks defense gene activation and phytoalexin accumulation without affecting ion fluxes, MAP kinase activation, cell viability and constitutive gene expression. Mimicking the oxidative burst in the absence of elicitor by addition of appropriate amounts of K02 to the medium of cultured parsley cells stimulates phytoalexin accumulation but no ion fluxes. In contrast, H202 either added directly to or generated in the culture medium by glucose and glucose oxidase does not stimulate any defense response. Our results demonstrate a causal relationship between early and late elicitor reactions, establish a sequence of signaling events from receptor-mediated activation of ion channels through MAP kinase activation, the oxidative burst, and defense gene activation to phytoalexin production, and suggest that within this signal transduction chain the superoxide anion radical rather than H202 triggers defense gene activation and phytoalexin accumulation.

5. What are some other elicitors besides those mentioned above?

1. Ethylene and Salicylic acid which were mentioned previously as a plant "hormones" may be carried long or short-distances in the plant and act as an amplifier of attack signal. There is recognition that other plant killers i.e.. ozone, droughts also induce ethylene production, thus this may be a generalized response to stress.

(January 24, 1997 issue of Science has a significant article documenting for the first time clear evidence that ethylene biosynthesis plays a direct, causal role in plant disease response. The article is "A Legume Ethylene-Insensitive Mutant Hyperinfected by Its Rhizobial Symbiont." R. Varma Penmetsa and Douglas R. Cook, Dept. of Plant Pathology and Microbiology, Crop Biotechnology Center, Texas A+M)

2. Exogenous elicitors besides the oligopeptide, Pep-13 mentioned above include chitin fragments, arachidonic acids and other components of the cell wall or cell membrane.  

3. As the plant cell itself is being digested it gives off endogenous elicitors including pectic fragments....

4. As mentioned above change in cellular redox potential due to changes in H+/ Ca++/K+ can themselves act as signals to surrounding cells/ tissues that the plant is under attack... 

5. As the plant produces defense related proteins ( these are specific proteins produced by plants when attacked; they role is not completely understood at this time... some are produced in the vacuole and other intracellularly; they have known microbial inhibition properties and may be proteinase inhibitors or proteinases and build up with infections immunity defense system; they may act additively or in synergy to enhance disease resistance or interact with other factors for disease resistance) and other metabolites

 

BIOSCIENCE COMPANY PIONEERS PLANT STIMULANT
Tucked away in a nondescript office park near Bothell, Wash., Eden Bioscience is growing a high-tech field of dreams amid the rows of tobacco, strawberry and tomato plants in its greenhouse laboratory.

Working with researchers at Cornell University, the company has pioneered an all-natural plant stimulant known as "Messenger" that delivers a triple benefit to food growers: It boosts the growth of a variety of crops. It helps block a number of common plant diseases. And it helps plants repel insects and nematodes.

Scientists have found that plants have a much greater capacity to ward off illnesses and bugs than previously thought. What's needed is a catalyst to unleash that extra fighting power.

Messenger does not alter the genetic makeup of the plants. Rather, using a naturally occurring protein as its active ingredient, Messenger acts as a health supplement. Eden calls it a plant "vaccine.

With Messenger's help, said Eden President and Chief Executive Officer Jerry Butler, "You're turning the right things on. Messenger is the first product for Eden, a privately held company that became one of the few agricultural biotech companies in the Northwest when it formed five years ago.

The Environmental Protection Agency is reviewing Messenger and is expected to approve it later this year or early next year.If the EPA gives the green light, Messenger could be used not only on tomato, tobacco and strawberry crops but also on wheat, cotton, soybeans and two dozen other crops. There are no sales projections for Messenger. But the total market for pest- and weed-fighting crop products worldwide is more than $18 billion.

But winning approval from the EPA is just half of Eden's battle. The company also must convince farmers that Messenger is better than anything already on the market. No other company is believed to have a natural crop treatment in development that does everything Messenger's harpin protein can do. There are chemical-based products on the horizon, however, that could pose competition.

"The farmers of the world are among the most difficult sells to make," Eden Chief Financial Officer Brad Powell said, noting growers are bombarded with so-called miracle crop-enhancement products that may or may not work in their own fields. "That's why we've taken a `We'll show you' approach to marketing," he said.

Eden has done about 300 field trials using Messenger on vegetable, fruit and other crops in the Southeastern, Midwestern and Western United States, as well as Mexico and China. The company has permission to treat up to 5,000 acres for demonstrations and tests. Field trials on crops in China have resulted in yield increases of 30 percent to 40 percent, Butler said. In the United States, where crop-management techniques are far more advanced, there have been yield improvements of 15 percent to 20 percent in many cases.

However, Butler is the first to say that Messenger is no magical plant elixir. It doesn't cure plant diseases. It

doesn't kill germs and insects. It simply helps plants resist attack and infection while stimulating growth. The company intends Messenger to be used in combination with standard chemicals for the best results. The active ingredient in Messenger is a protein called harpin, found in bacteria that cause the pear and apple disease fire blight.The scientists discovered that plants are hypersensitive to a form of the protein that apparently triggers the plants' defenses, a reaction that usually occurs within five minutes.

And as a bonus, researchers at Cornell were amazed to find, harpin makes plants grow faster and larger and discourages insects from living on a plant's surface. "It was a surprise and still is a mystery," Wei said. "It still is unknown how that happens."

Eden has cloned the harpin gene and mass produces the protein in fermentation tanks at its headquarters in Bothell. The protein is dried, then mixed with starch to produce Messenger. Two grams of harpin, which when combined with the starch fits into a freezer-bag-sized pouch, can be mixed with water to spray one acre of crops. It leaves no detectable residue, and the protein itself decomposes almost immediately after the application. There are no known harmful side effects to the plants, soil or animals that consume products from treated crops.

In scientific terms, Messenger's effect is systemic. Once it's sprayed on the exposed part of a plant, the Messenger protein activates an immune and growth response in every section of the plant, from root to stem to leaf. "It goes a little into overdrive," Wright says of the plant treated with harpin.

He sees Messenger, if it's priced right, as a potentially good thing for U.S. farmers at a time when crop prices are sagging. Messenger could reduce the need for other chemical-based crop treatments, such as pesticides, thereby cutting overall production costs.

In a sense, agriculture is entering its own brave new world. Since the mid-1990s, the agricultural biotech industry has exploded, with a number of small companies popping up to develop healthier crops that produce larger and better quality yields. Seed and crop-protection companies are currently spending about $900 million on research into biotechnology-based products, said Ken Moonie, a consultant with Verdant Partners, an investment banking firm that specializes in seed and plant biotechnology. That's up from about $150 million.

Source: The Seattle Times

Knight Ridder/Tribune Business News

III. How do fungi and other microbes protect themselves from their host's defense system?

A. some may produce enzymes which inactivate phytoalexins. The abstract below indicates possible pathways to resistance by fungi towards plant defense systems.....

Phytoalexin Detoxification Genes and Gene Products: Implications for the Evolution of Host Specific Traits for Pathogenicity

H.D. VanEtten, Department of Plant Pathology

Production of phytoalexins by plants is believed to function as a mechanism of disease resistance. Many fungal pea pathogens have the ability to detoxify the pea phytoalexin pisatin via demethylation. This detoxification may be a means to circumvent a phytoalexin-based resistance mechanism. The detoxifying enzyme, pisatin demethylase, has been studied most thoroughly in Nectria haematococca. We have completed an examination of the induction of pisatin demethylating activity in whole cells and the biochemical properties of pisatin demethylase in microsomal preparations from the pea pathogens Fusarium oxysporum f. sp. pisi, Mycosphaerella pinodes, and Ascochyta pisi and compared these properties to those of the enzyme produced by N. haematococca. All of the enzymes are cytochrome P450s, based on cofactor requirements, and their inhibition by carbon monoxide, cytochrome P450 inhibitors and antibodies to NADPH cytochrome P450 reductase. In addition, all of the enzymes were selectively induced by pisatin, had a low Km on pisatin and a high degree of specificity towards pisatin as a substrate suggesting the presence in each pathogen of a specific cytochrome P450 for detoxifying pisatin. However, since the pisatin demethylases differed in their pattern of sensitivity to P450 inhibitors and displayed other minor biochemical differences, these fungi may have independently evolved specific cytochrome P450s to detoxify the phytoalexin produced by a common host.

Ralph L. Nicholson: We are using the conidial mucilage of Colletotrichum graminicola as a model system. The mucilage acts as an antidesiccant and maintains spore viability during periods of dryness. It also contains several enzymes and proline-rich proteins that function as protectants against plant phenolics. This system is providing a tool for understanding the basis of survival as well as factors essential to virulence of the pathogen such as the process of adhesion.

B.Virulent fungal strains suppress hosts ability to produce phytoalexin in the first place

C. Produce compounds such as oxalic acid which chelates Ca++. Calcium may be critical for the ion fluxing reaction involved in the first series of defense reactions ( see above) or many other critical metabolic pathways.

Go on to the next page how plant use these compounds to compete with one another or to more on medicinal botany or return to the original defense page.....

Iglich/botany01