While rice is used in cultural aspects it also affects a region environment.

Due to rice’s already

high national yield, further increase in rice yield requires the development and use of novel

technologies. However, there are limited land area for expansion, there is salinization of rice

fields in semi-arid zones, increasing nutrient deficiency due to decreasing use of organic

fertilizers and increasing crop intensification, low temperature in Northern parts, flood in low-

lying areas, and various weeds, insects, and diseases (18).

Around the world The International Genome Sequencing Project (IRGSP) has been sequencing

monocots since it started with the first meeting in Singapore established by the Rockefeller

Foundation and led by The Rice Genome Program of Japan (RGP)

(9). Interested countries agreed to sequence Oryza sativa ssp. Japonica c.v. Nipponbare with the

use of large insert BAC (developed at CUGI) and PAC (RGP) libraries. Additionally, there are

about 35, 000 EST’s (Expressed Sequence Tags) from various rice cDNA libraries exposed to M.

grisea under different conditions deposited into GenBank and is providing affordable public

access to the clone resource (10).

The main way to rectify the problem of M. griseais to make the plant resistant to the pathogen by

altering the plant’s genetic make-up or discovering a way of stopping the pathogen before it

spreads to the crop. In order to do such, insight must be gained by understanding the host-

pathogen interaction by characterizing the genes of the fungus. In this project, the method for

identifying important genes is through using Agrobacterium tumefaciens mediated

transformation to randomly mutate genes in the genome of M. grisea.

Magnaporthe grisea pathogenicity genes can be obtained through insertional mutagenesis (12

and 13). Mutagenesis allows for the identification, characterization, and elucidation of the

mechanisms of physical, chemical, and biological agents capable of producing genetic change in

living organisms and the consequences of those changes. A mutational analysis for the

pathogenicity of the rice blast fungus was conducted (12) using restriction enzyme-mediated

integration (REMI) for insight on its ability to infect plants. Exactly 5,538 transformants were

screened and out of those mutants 27 exhibited a reproducible pathogenicity defect while 18 of

those mutants contained mutations that consegrated with the hygromycin resistance marker.

Eight of the mutants were used to clone seven PTH genes that play a role in pathogencity on

barley, rice, and weeping lovegrass. Two of the cloned mutants were characterized as having the

same gene, PTH2. This implied that nonrandom insertion of the transforming DNA. Random and

targeted insertions of genes via transformation are two very effective ways to study the infection

mechanisms of fungal pathogens. This allows scientist to isolate the mutants that exhibited

altered virulence. Employing the binary vector pBHt2 (carries the bacterial hygromycin B

phosphotransferase gene) led to the production of 500 to > 1,000 hygromycin B-resistant

transformants. A genomic Southern Blot analysis revealed over 60% of the transformants held a

single T-DNA insertion in their genome (2).

The pathogen life cycle of Magnaporthe grisea involves a minimal developing sequence (12). It

begins with the deposition of a conidium on the plant surface and then the germination of the

conidium to form a germ tube. Differentiation of the germ tube into a specialized infection

structure called an appressorium. Through the appressorium the fungus is able to penetrate the

plant’s leaf surface by the melanized appressorium, accumulation of hydrostatic pressure, and

development of a penetration peg. Differentiation of the penetration peg occurs making it into a
secondary hypha that then grows throughout the invaded plant cell. The fungi then escapes from

the first infected plant cell and transfers itself to the surrounding tissue. Aerial conidiophores,

and conidia are then produced and the final step is for the releasing of the conidia into the same

or surrounding plants were the life cycle is repeated. These cycles can repeat over a dozen times

a growing season spreading millions of conidia around a rice field.

(Figure 3 Appressorria formation and infection)

Magnaporthe grisea, the heterothallic ascomycete, has the potential for sexual and asexual

production (14). Data from mating type, fertility essays, and genotypic diversity strongly suggest

that the pathogen is asexual in field conditions. However, parasexual recombination is not yet

ruled out. Chromosome length polymorphisms and tranocations may stop successful meiosis

from occurring in most populations. Pathogens of millets and various grasses growing with rice

appear to be largely genetically isolated.

Formation of he appressorium is critical for M. griseato be able to incite disease. The

appressorium is a structure employed by fungal pathogens to press against and attach to the plant

surface in preparation for infection. It is a required tool for the fungus to infect the host. For the

formation of the appressorium, there needs to be the cAMP-dependent protein kinase, which is

necessary for the infection-related morphogenesis and pathogenesis in a phytopathogenic fungus

(1). The gene encoding the catalytic subunit of the cAMP-dependent protein kinase was cloned,

sequenced, and disrupted. The result was two transformants that were unable to form

appressorium. Various environmental cues such as derivatives of the cuticle from the leaf

surface, wax components, and chemical signals stimulate the growth of appressoria. It is believed

that perception of environmental signals, mediated by specific receptors, are likely to signal

transduction pathways that lead to the differentiation of the appressorium (4).These pathways are

very important to the appressorium and for the research of how it works. Compelling evidence

suggests that a particular Resistance gene is important in the preferential utilization of either

signaling components and define two disease resistant pathways (5).