FIELD OF THE INVENTION

The present invention relates to the development of transgenic plants via Agrobacterium that are resistant to both abiotic and biotic stress. The invention more specifically relates to co-transfer and expression of chitinase and Mn-SOD genes in pearl millet for conferring resistance to fungal attacks including Downy Mildew and drought tolerance.

BACKGROUND OF THE INVENTION

Most agricultural and horticultural crops are under a constant threat due to various pathogens. To protect the crops from significant losses from them including fungal disease, the crops and sometimes the soil in which the crops are grown are periodically treated with large amounts of fungicides. These fungicides form a heavy burden on costs of crop growing, and more importantly on the environment and the growers. Moreover the treatment is very labour intensive. Therefore, there is a need for less costly and safer methods to protect plants from abiotic stress and fungal attack that, preferably, are devoid of the need for repeated human involvement.

Environmental stress in a broad sense is a restriction placed on living organisms by nature. The definition of environmental stress in plant science is a set of physical and chemical factors affecting the environment consequently disturbing plant growth. This stress could occur due to variant temperatures be it high or low, insufficient water supply, ultraviolet radiation and emission of pollutant gases. The study of environmental stress in plant life is significant on account of the fact that world over agricultural productivity has been greatly restricted by it and the need to withstand this kind of environmental stress is a prerequisite when studying plant life. The production of reactive oxygen species in cells is an inevitable restriction on aerobic life and use is made of the oxidative atmosphere for yielding energy at a high efficiency. In so far as the metabolism under non stressful conditions is concerned, reactive oxygen species is always produced. The reactive oxygen species is produced in the cells for biosynthesis, cell defence, intra and intercellular signalling.
Hence, reactive species of oxygen is both, indispensable as well as toxic to life.

It has been observed that reactive oxygen species plays a crucial role in the impairment of cellular functions due to environmental stress viz, increase in the productive of reactive oxygen species and production of oxidised target molecules under stress, decrease in the antioxidant levels or contents under stress, increased expression of the genes for anti-oxidative functions by stress, positive co-relation between the scavenging capacity for reactive oxygen species and tolerance towards stress, cross tolerance between oxidative stress and other stress.

Oxidative damage caused by reactive oxygen species can be induced by two principal mechanisms viz, an enhanced production of reactive oxygen species or by an inhibition of the scavenging systems for them. The damage proliferates production of a highly reactive hydroxyl radical and the subsequent reactions like bleaching of pigments and accumulation of oxidised lipids are apparent, these being the final symptoms of oxidative damage observed in dying cells.

Superoxide is a commonly encountered mediate of oxygen reduction. It is extremely toxic to cells since it attacks unsaturated fatty acid components of membrane lipids thereby damaging the membrane structure. Aerobic cells detoxify super oxide by the action of super oxide dismutases, metal containing enzymes that convert the superoxide radical into hydrogen peroxide and molecular oxygen. The hydrogen peroxide later converted by catalase into water and molecular oxygen.

There are three types of super oxide dismutase (SOD), copper/zinc containing SOD (CuZnSOD), manganese containing SOD (MnSOD) and iron containing SOD (FeSOD). In prokaryotic organisms MnSOD is inducible under conditions of high oxygen concentration and by O2.

In eukaryotes, the MnSOD is a nuclear encoded protein that scavenges superoxide radicals in the mitochondrial matrix. By targeting this enzyme to the chloroplast where the generation of superoxide radicals is high during stress conditions, the capacity to scavenge any radical that may be produced can be increased. In an attempt to improve stress tolerance ofplants including pearl millet plants.

Plants are provided with improved resistance against pathogenic fungi. They are genetically transformed with one or more polynucleotides which essentially comprise one or more genes encoding plant chitinases and P-l,3-glucanases. Preferred are the intracellular forms of the said hydrolytic enzymes, especially preferred are those forms which are targeted to the apoplastic space of the plant by virtue of the modification of the genes encoding the said enzymes. Particularly preferred are plants exhibiting a relative overexpression of at least one gene encoding a chitinase and one gene encoding a P-1,3-glucanase. A group of induced genes encodes proteins which accumulate both inside the cells and in the apoplastic space. Among these proteins are hydrolytic enzymes such as chitinases and glucanases. After a necrotic infection these enzymes can often be found throughout the plant, including the non-infected parts, in higher concentrations than before infection. Increased synthesis of these enzymes appears to be induced also by microbial elicitors, usually fungal cell wall preparations (Darvill & Albersheim, 1984; Toppan & Esquerre-Tugaye, 1984; Mauch et al., 1984; Chappel et al., 1984; Kombrink & Hahlbrock, 1986; Hedrick et al, 1988). The cell walls of fungi are known to consist of a number of different carbohydrate polymers. Most fungi, with the exception of the Oomycetes, contain considerable amounts of chitin. Chitin is a polymer of N-acetyl glucosamine molecules which are coupled via P-1,4 linkages and, in fungal cell walls, are often associated with P-l,3/p-l,6 glucan, polymers of glucose with p-1,3 and P-1,6 linkages. Fungi from the group of Zygomycetes do not contain glucans with P-1,3 and P-1,6 linkages, while in most of the Oomycetes the glucans are associated with cellulose (for an overview, vide: Wessels and Sietsma, 1981).
It has been known for a long time that isolated cell walls of fungi can be degraded in vitro by plant extracts (Hilborn & Farr, 1959; Wargo, 1975; Young & Pegg, 1982) and also by chitinase and P-l,3-glucanase preparations from microbial origin (Skujins et al., 1965; Hunsley & Burnett, 1970; Jones et al., 1974).

A purified P-l,3-glucanase from soybean (Keen & Yoshikawa, 1983), as well as a purified chitinase from bean (Boiler et al., 1983) have also been shown to be capable of degrading isolated cell walls of fungi in vitro. When pea chitinase and p-l,3-glucanase were tested on isolated cell walls of Fusarium solani, both appeared to be active; in combination they appeared to work synergistically (Mauch et al., 1988b).

It is not known whether these hydrolytic enzymes can degrade the polymer compounds in cell walls of living fungi effectively, if at all.
Inhibition of Fungal Growth on Synthetic Media by Chitinases from plant origin has also been observed. Some chitinases and glucanases of plant origin are capable of inhibiting the growth of fungi on synthetic media. A combination of chitinase and (3-1,3-glucanase, both purified from pea pods, do inhibit the growth of some fungi on agar plates. Little is known about the effect of hydrolytic enzymes on fungi in the biotrope, i.e. in the soil or on plant leaves, and although some of these enzymes are putative candidates for a role in fungal resistance, evidently, not all chitinases and glucanases have activity against living fungi.
Possibly, the stage and site of infection at which hydrolytic enzymes come into contact with the invading fungus may be of great importance.

OCCURRENCE OF CHITINASES IN PLANTS

As far as known, chitinases occur in most if not all plant species; both in monocotyledonous and dicotyledonous plants. At least two classes of chitinases can be discriminated: intracellular and extracellular. Chitinase genes of one particular class appears to be encoded by gene families.

NATURAL EXPRESSION OF CHITINASE GENES IN PLANTS

Chitinase genes are known to be expressed in plants both constitutively and in a strictly regulated fashion.

Chitinases are constitutively synthesised in roots of tobacco plants (Felix and Meins, 1986, Shinshi et al., 1987,; Memelink et al., 1987, 1989). Nevertheless tobacco plants are not resistant to infection of Phytophthora parasitica var. nicotianae (a root pathogen of tobacco). However, resistance against this pathogen can be induced in tobacco plants,
following inoculation with TMV (Mclntyre & Dodds, 1979). This suggests that a complex of yet unknown factors other than, or in addition to, chitinases and glucanases, may be involved in fungal resistance.

On the other hand, plant species are known which seem to be resistant to fungal infection, although no significant increase in the levels of chitinases or glucanases can be observed. For instance, in tomato a compatible interaction with the fungus Phytophthora infestans causes a systemic resistance (Christ & Mosinger, 1989), i.e. a resistance to infection throughout the whole plant, although chitinases or glucanases cannot be detected in such leaves (Fischer et al., 1989). Apparently there is no clear correlation between expression of the genes encoding hydrolytic enzymes and fungal resistance.

In addition to these observations, some chitinases exhibit a regulated expression pattern which does not immediately suggest a correlation with fungal resistance.

For example, genes encoding chitinases are known to be expressed in a developmentally regulated manner in, inter alia, tobacco flowers (Lotan et al., 1989). Glucanases are known to occur in large quantities in seedlings of barley (Swegle et al., 1989; Woodward & Fincher, 1982; Hoj et al., 1988, 1989).

Plants contain at least two classes of chitinases: extracellular and intracellular. The expression of the genes encoding the said hydrolytic enzymes is not constitutive, at least not in all tissues, but is among other things regulated in a developmental or tissue-specific fashion. However, the expression of the genes can also be induced under certain stress-conditions, such as an infection by a necrotisizing pathogen. In most cases, induction of the synthesis of chitinases and p-1,3-glucanases is accompanied by the induction of resistance against a broad range of pathogens, including phytopathogenic fungi. Whether there is a causal relation between fungal resistance and expression of the genes encoding hydrolytic enzymes is not clear.

Cell walls of phytopathogenic fungi contain glucans and often a certain amount of chitin. These carbohydrate polymers are substrates for glucanases and chitinases, respectively. It is attractive to hypothesize that both hydrolytic enzymes are responsible for the observed
resistance. However, this is far from obvious, in view of many observations which are clearly in conflict with this hypothesis.

Hence, it is still far from clear whether hydrolytic enzymes have a significant role in fungal resistance, or, when they appear to have so, how substantial their role in fungal resistance is. It seems at least doubtful that any chitinase can confer broad range protection of plants against phytopathogenic fungi.

Generally, it is even questionable if chitinases and glucanases by themselves are capable of providing sufficient protection against a broad range of plant pathogenic fungi.

There is still little basic understanding of the role of hydrolytic enzymes in the complex process of acquiring (induced) fungal resistance. However, there is a need for a method to effectively protect plants against (a broad range) of phytopathogenic fungi, by means of genetic modification.

Any plant species or variety that is subject to some form of fungal attack may be transformed with one or more genetic constructs according to the invention in order to decrease the rate of infectivity and/or the effects of such attack.

BRIEF SUMMARY OF INVENTION

The aim of the present invention is to provide plants that are drought tolerant and also resistant to biotic stess i.e., fungal attack, with and p-l,3-glucanase genes. Thereto, plants are genetically transformed via Agrobacterium-mediated transformation by introducing into the genome of the said plants Mn-SOD and chitinase DNA-constructs that is under the control of a promoter which is not naturally associated with that gene.

More in particular the invention provides plants having improved resistance to fungal attack, by virtue of the expression of at least one recombinant DNA-construct that comprises a DNA-sequence, encoding at least-one intracellular plant chitinase, which is modified such that the intracellular chitinase becomes secreted into the apoplastic space.

Furthermore the invention provides plants having improved resistance to abiotic stress such as drought by virtue of the expression of at least one recombinant DNA-construct that comprises a DNA-sequence, encoding MnSOD.

The invention provides plants exhibiting a more effective protection against fungal attack due to the expression a gene encoding a chitinase, preferably an intracellular chitinase, and a gene encoding a glucanase, under the control of a promoter that allows suitably strong expression, in one or more tissues. The invention provides plants constitutively expressing a gene encoding an intracellular chitinase of plant origin which is targeted to the apoplastic space and, additionally, one or more genes encoding a hydrolytic enzyme from the group consisting of intracellular chitinases, extracellular chitinases, intracellular glucanases and extracellular glucanases.

Of these, genes encoding the intracellular forms of the mentioned plant hydrolytic enzymes are particularly preferred. Further embodiments of the invention are the recombinant DNA molecules, comprising one or more plant expressible DNA sequences encoding at least one intracellular chitinase of plant origin which is modified to achieve targeting of the chitinase to the intercellular space, and, if desired additional DNA sequences encoding one or more hydrolytic enzymes selected from the group consisting of extracellular chitinases, intracellular glucanases and extracellular glucanases.

A construct containing Mn-SOD and chitinase gene will be co-transferred with a construct containing a selectable marker into Pearl millet using Agrobacterium mediated transformation.
Plants are provided with improved resistance against pathogenic fungi. They are genetically transformed with one or more polynucleotide that essentially comprise one or more genes encoding plant Mn-SOD and chitinases.

Example 1

METHOD OF AGROBACTERIUM MEDIATED TRANSFORMATION AND REGENERATION

Sterilize the seeds of pearl millet (variety 843B) with 50% bleach for 5 minutes, then with 0.1% HgCl2 for 7 minutes and then with 70% alcohol for a minute and finally wash with autoclaved deionized water.

1. Place the surface sterilized seeds on MS medium containing 3ppm of 2,4-D and 0.3ppm of BAP for callusing.

2. Subculture the calli after 21 days onto callusing medium (MS medium containing 3ppm of 2,4-D and 0.3ppm of BAP).

3. After 21 days, transfer the calli to fresh MS-medium containing 3ppm of 2,4-D and 0.3ppmofBAPl.

4. After 3 days, remove the watery callus and subculture the embryoids of calli to MS-medium containing 3ppm of 2,4-D.

5. Calli were placed for Agrobacterium infection for one hour and were transferred to co cultivation medium (supplemented with 100 uM acetosyringone) which were incubated at 28 °C for 3 days in the bacteriological incubator.

6. Wash the infected calli twice with 250 ppm cefatoxime followed by wash with sterilized water thrice and transfer them to MS medium with 3 ppm 2,4-D and 0.3 ppm BAP.

7. After 15 hours of incubation in dark, transfer the calli to MS-medium with 3ppm of 2,4-D and 30ug of hygromycin per litre of medium.

8. After 21 days, subculture the proliferating calli to MS-medium containing lppm of 2,4-D and 30^i g of hygromycin per litre of medium.

9. After 21 days, subculture the proliferating calli to pearl millet regeneration medium (viz.,MS-medium containing 5ppm of BAP and 0.5ppm of NAA per litre of medium)

10. After shoot differentiation of calli, subculture the regenerants to l/2 MS-medium for rooting.

11. After the rooting of regenerants, harden them in plain water for 2 days and transfer them to pots in green house.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA sequence of a chitinase gene (SEQ ID NO 1)

FIG. 2 shows the DNA sequence of an MnSOD (SEQ ID NO 2)

We claim

1. A method of enhancing plant resistance to a fungus along with drought tolerance,
the method comprising: a) introducing in to a plant a recombinant gene encoding a chitinase set forth in SEQ ID No. 1; and a polynucleotide encoding MnSOD set forth in SEQ ID No.2; b) selecting a plant with enhanced resistance to pathogens that are also drought tolerant.

2. The plant according to claim 1, wherein said chitinase gene is operably linked to a
promoter.

3. The plant according to claim 1, wherein said MnSOD gene is operably linked to a
promoter.

4. A transformed plant of claim 1 said transformed plant containing DNA sequences
encoding for chitinase set forth in SEQ ID No. 1 and DNA sequences encoding for MnSOD set forth in SEQ ID No. 2.

5. The transgenic plant of claim 4 wherein the plant is selected from the group consisting of maize, soyabean, jatropha, pongamia, canola, castor, groimdnut and coconut, pearl millet, sorghum, sugarcane.

6. The transformed plant of claim 4, wherein the transgenic plant is a pearl millet plant.