6. arroz resistente

8/11/2019 6. Arroz Resistente

1/6

Rice is arguably the worlds most importantfood. Almost two billion peopleone thirdof the worlds populationdepend primar-

ily on rice for basic nourishment. Rice fields covermore than 360 million acres of land around theglobe and yield 560 million tons of grain every year.But farmers plant much more rice than they harvest,because insects, bacteria, viruses and fungi often claima substantial portion of each crop. One of the mostdevastating of these pestilences is blight, caused bybacteria common throughout Asia and Africa.

These bacteriaXanthomonas oryzae pv. oryzae(known as Xoo)spread rapidly from rice plant torice plant and from field to field in water droplets. In-fected leaves develop lesions, yellow and wilt in a

matter of days. In severely infected fields, bacterialblight can wipe out half of a farmers rice crop.

And yet rice plants possess an amazing assortmentof genes that offer protection from a host of diseases,including bacterial blight. The farmers predicamentis that no single variety has every gene and that allplants are vulnerable to some diseases more than toothers. Breeders have exploited disease-resistancegenes in rice for nearly a century, redistributing thisgenetic wealth from hardy species to agriculturallyuseful varieties. But conventional breeding is pain-staking and time-consuming; often a decade or moreis needed to produce desired traits.

With the advent of genetic engineering, we are nowable to introduce isolated disease-resistance genes di-rectly into rice plants, trimming years from the timerequired to develop a useful variety. My colleaguesand I recently cloned the first such disease-resistance

RICE PLANTS are subject to many destructive diseases,including bacterial blight, which causes devastating leafdamage and reduces yield (left). Water droplets carrythe bacteria into leaf wounds; yellow lesions develop oninfected leaves in days. If infected while very young, theentire plant may succumb. Certain plants, however,

have a genetic resistance to blight (right).

Making RiceDisease-ResistantFor the first time, scientists haveused genetic engineering to protect

this essential crop from disease

by Pamela C. Ronald

Copyright 1997 Scientific American, Inc.


2/6

Scientific American November 1997 101

gene from ricea gene that protects against commonforms of bacterial blight. We have used this gene togenerate the worlds first transgenic disease-resistantrice plants. These new varieties of rice have tremen-dous potential to aid farmers around the world.

Gene Hunt

The story of the first disease-resistance gene cloned

from rice begins in the developing world. Oryzalongistaminata is a wild species of rice native to Mali,useless as a crop plant because of poor taste and lowyields but quite hardy when it comes to resisting bac-terial blight. In 1977 researchers in India evaluatedthis plant for resistance to various strains of Xoo andfound it could withstand every type they tested.

A year later Gurdev S. Khush and his co-workersat the International Rice Research Institute (IRRI) inthe Philippines began work with O. longistaminata.Their goal was to transfer blight resistance from thewild rice to a cultivated variety, using conventionaltechniques. Twelve years of intensive breeding yield-ed one such resistant variety as well as the knowledgethat the resistance was conferred by a small region ofa single chromosome, perhaps even a single gene,which they named Xa21.

In 1990, just as they were reaping the intellectualrewards of this labor, I became a postdoctoral fellowat Cornell University. In an effort to learn about themechanism of bacterial blight resistance, I decided totry to clone Xa21 from the IRRI variety. A gene suchas Xa21 is a short stretch of genetic material (DNA)that encodes a protein and often, therefore, a trait(such as blight resistance). Although we frequentlystudy traits in whole organisms, we cannot easilystudy single genes in the context of an organisms to-

tal genetic content, or genome. Nor can we accom-plish genetic engineering without isolated genes. Thesolution is to generate accessible copies of individualgenes in the process known as cloning.

To clone Xa21, I needed to identify the precise re-gion of the rice genome that bears this gene, transferthis bit of DNA into bacteria where it could be easilycopied, insert these copies into susceptible rice plantsand then prove that the inserted DNA made theseplants resistant to blight. When I began my effort, noone had yet succeeded in cloning a disease-resistancegene from any plant, although many such genes wereknown and used in traditional breeding. At that time,

the barriers to genetic engineering of disease resis-tance fell into two categories: problems in finding thegenes and problems in moving them around.

Finding a gene in a genome is a lot like the prover-bial search in the haystack, and the genomes of mostplants are particularly huge haystacks. As a rule ofthumb, it is harder to locate specific genes in large ge-nomes than in smaller ones; large genomes are alsotough to manipulate. A simple yardstick is the tinygenome of the bacterium Escherichia coli, from whichwe can isolate genes quite easily. By this measure therice genome is largealmost 100 times the size of theE. coli genome. A gene hunt in rice is challenging: my

own search for Xa21 would eventually require sever-RICHARDJ

ONES



3/6

al years and many sophisticated tech-niques. Still, I was fortunate; research-ers seeking to isolate genes from certainother grains face even bigger barriers.The genome of wheat, for example, isalmost 3,500 times the size of the E.coli genome and fully five times the sizeof the human genome. Cloning a genefrom grains such as rice and wheat is

extremely difficult without some priorknowledge of the genes location or se-quence (think of trying to find a friendshouse in New York City or Tokyo with-out an address or description).

In 1990 I felt the time was right forcloning genes from rice, because pio-neering work led by Steven D. Tanksleyand Susan R. McCouch, also at Cor-nell, had just produced a key develop-ment: a map to guide my exploration ofthe vast rice genome. The type ofcloning I used is known as map-basedcloning, and as the name implies, it re-quires some knowledge of the locationof various landmarks, or markers, inthe DNA. The genetic map constructedby the Cornell group showed the loca-tions of hundreds of useful markers onthe 12 rice chromosomes.

Over a period of a few years, first atCornell and later at the University ofCalifornia at Davis, my colleagues andI used this map to track down Xa21.During our search, we examined morethan 1,000 rice plants to see how oftenthe known DNA markers showed up in

conjunction with resistance to bacterialblight. This strategy takes advantage ofa certain amount of chromosomal swap-ping and rearranging that goes on dur-ing sexual reproduction: the closer twosites on a chromosome are, the less like-ly they are to be separated from eachother during this process of recombina-tion. In our case, the more often wesaw resistance passed to progeny alongwith a given marker, the closer the re-sistance gene must lie to that marker.

By sheer luck, the first chromosomallandmark that my group and I identifiedas lying very close to Xa21 turned outto be incredibly useful. One weekend inMay 1994, two years after I set up myown laboratory at Davis, I discoveredthat the sequence of the marker DNAwas similar to that of several disease-re-sistance genes recently cloned from to-

bacco, tomato, flax and a mustard plant.Alone in the lab that Sunday morning, Icalled my longtime friend and colleague

John Salmeron of the University of Cal-ifornia at Berkeley and asked him tocompare my sequence more carefullywith his tomato disease-resistance gene.We were thrilled to find very strong sim-ilarities among genes from such differ-ent plants. I felt confident that I wassearching in the right neighborhood.

My group and I spent the next yearcloning candidate Xa21 genes and pre-paring to insert them into other riceplants. We knew that the crucial testwould come when we transferred ourisolated rice DNA into a plant normallysusceptible to Xoo. If we had cloned theright gene, the resulting transgenic plantswould be resistant to bacterial blight.We were anxious to begin these experi-ments, but we faced an undeniable ob-stacle: we had no experience introducinggenes into rice cells. And at that time,only a very few labs in the world wereable to carry out this process, calledtransformation, in rice.

Under the Gun

This problem of transferring genesinto plant cells is the second greathurdle in engineering disease resistance.Many types of plant cells, including rice,are refractory to taking up extraneousDNA. The breakthrough came in 1987,when John C. Sanford of Cornell devel-oped a gun that shoots microscopic par-ticles into intact cells [see TransgenicCrops, by Charles S. Gasser and Rob-

ert T. Fraley; Scientific American,June 1992]. Sanfords early versionswere propelled by a gunpowder charge;later models are helium-driven and firepellets made of gold. These pellets, whichare less than a hundredth of a millimeterin diameter, can be coated with DNAthat they then carry directly into cells.

Researchers did not use this techniquein rice until 1991; when we were readyto test our Xa21 clone, the Internation-al Laboratory for Tropical AgriculturalBiotechnology (ILTAB) was one of the

facilities doing so routinely. It is conve-

Making Rice Disease-Resistant102 Scientific American November 1997

TRADITIONAL BREEDING has beenused for years to produce disease-resistantrice. Pollen from a resistant plant fertilizesa susceptible plant that also has desirablecharacteristicsit produces high yields ofgrain, for instance, or tastes good. Prog-eny inherit a random mixture of geneticmaterial from both parents (colored bars).Resistant progeny are crossed again withthe susceptible plant, endowing offspringwith more of that parents valuable traits.Selection of resistant progeny at each crossensures the continuing presence of the re-

sistance genein this case,Xa21.

RESISTANTPLANT

SUSCEPTIBLEPLANT

SELECTION OFRESISTANTPROGENY

Xa21

Xa21

RESISTANTPLANT

RICHARDJONES



4/6

niently located in California and, to ourdelight, agreed to help us.

Researchers at ILTAB used the gun totransform our cloned DNA into ricecells of the variety Taipei 309. This is anold variety that is no longer grown, butwe chose it because it is easily trans-formed and susceptible to Xoo. We grew1,500 plants from the transformed cells;

each plant had a bit of cloned DNA inevery cell. When our plants were sixweeks old, it was finally time to test forresistance to bacterial blight.

We exposed each of our transgenicplants to Xoo by trimming their leaveswith scissors dipped in a bacterial sus-pension. Ten days later we examined theplants for lesions caused by the bacte-ria. We found that of the original 1,500transgenic plants, 50 plants were highlyresistant to infection with Xoo: eachhad lesions between 75 and 90 percentshorter than those in the original sus-ceptible plants. In these 50 plants, thetransformed piece of DNA containedan intact blight-resistance gene.

Pice de Rsistance

We had succeeded in cloning Xa21.Subsequently, we showed thatXa21 was passed on to the next genera-tion through self-fertilization, givingrise to seedlings that were also resistantto bacterial blight. We challenged ourtransgenic plants with 31 different Xoo

strains from eight countries spanningAsia and as far flung as Colombia. Theplants resisted infection by 29 of thesestrains, exactly replicating the disease-resistance profile of their wild Africanpredecessor. For the first time, we wereable to engineer rice for resistance tobacterial blight.

Our current goal is to insert Xa21into varieties that, unlike Taipei 309,are agriculturally important. In collabo-ration with ILTAB, we have successfully

Making Rice Disease-Resistant Scientific American November 1997 103

GENETIC ENGINEERING of diseaseresistance requires isolation of individualgenes that confer resistance. To isolateXa21, the author determined its approxi-mate location on rice chromosome 11 (a)and then generated pieces of DNA thatspanned the region (b). Next, the DNAwas copied in bacteria (c), and the repli-cas were placed on gold pellets that wereshot into cells of disease-susceptible riceplants (d). Some cells took up the foreignDNA but not the gene for resistance (e); afew cells, however, did receive intactXa21

and grew into resistant plants ( f).

a

CHROMOSOME 11

BACTERIALCELLS

RICE CELLNUCLEUS

GUN

GOLDPELLETSPELLET COATED

WITH DNACONTAININGXa21

RICE CELLS

TRANSFORMEDPLANTS

INFECTIONWITHXoo

SUSCEPTIBLE PLANTS RESISTANT PLANT

Xa21

b

c

d

e f



5/6

introduced Xa21 into twopopular varietiesIR64 andIR72which are grown onabout 22 million acres inAsia and Africa. Our ongo-ing studies show that thetransgenic plants are blight-resistant. And recently wehave also engineered resis-

tance in Ming Hui 63, a va-riety of rice widely grownin China.

With these exciting re-sults in hand, I have sentXa21 to scores of scientiststhroughout Europe, Africa,Asia and the U.S., with theobjective of introducingbacterial blight resistanceinto locally important ricevarieties. Because growingconditions vary greatlyfrom place to place, farm-ers often prefer to plant avariety of rice that is welladapted to their particularregion. These varieties pos-sess valuable traits such asdrought resistance, coldtolerance, short stature (forwind resistance) or resis-tance to indigenous pestsand diseases. The genetical-ly engineered versions willbe identical to the originalplants except for the addi-

tion of the single clonedgene conferring resistanceto bacterial blight.

Once we have generatedthese new varieties, we need to field-testthe plants for yield, taste and hardinessto establish that the useful traits of theoriginal varieties remain unchanged. Inthe next few years, researchers in Cali-fornia, Asia and Africa will field-testtransgenic rice containing Xa21. If theselines perform as well as locally adaptedvarieties, national breeding programs

will distribute seed to farmers in devel-oping countries. Because the disease-re-sistance transgene is passed on to pro-geny, farmers can grow their own seedfor the next season.

Crops of the Future

Compared with conventional breed-ing, genetic engineering is quickand flexible: we can shuttle individualcloned genes between plants in a matterof months. Donor and recipient need

not be compatible for breeding; we can

share genes among disparate species,even among different crops.

Thus, scientists should be able to har-ness cloned resistance genes to controldisease in many crops besides rice. Spe-cies of Xanthomonas that cause blight,for example, infect virtually all cropplants. In Florida, 99 percent of the cit-rus crop is susceptible, and growers

must closely monitor bacterial infec-tions to prevent epidemics. In the mid-1980s more than 20 million orangetrees were burned to thwart a suspectedoutbreak of this disease. State and fed-eral governments spent more than $40million on eradication alone, and hun-dreds of growers lost even more in un-realized produce. Scientists may some-day be able to protect citrus and otherlucrative crops by manipulating the ricebacterial blight-resistance genes andtransferring them into susceptible species.

Genetic engineering may also help us

cope with the problems anydisease-resistant plant facesonce it is in the field. In par-ticular, pathogens may mu-tate and overcome the pro-tection a given resistancegene confers. Breeders musttherefore continually iden-tify and introduce useful

genes in order to minimizesusceptibility to disease,whether through conven-tional methods or geneticengineering. Fortunately,many resistance genes areknown and are ripe forcloning. Combinations ofthese genes may further en-hance disease resistance,much the same way thatcombinations of antibioticsor antiviral drugs combatmicrobes such as tuberculo-sis bacteria and humanimmunodeficiency virus.

We also hope to incorpo-rate resistance to more thanone pathogen in a singletransgenic line. In some in-stances, farmers cannot userice varieties bred for resis-tance to bacterial blight,because they lack resistanceto other pathogens andpests. The most serious ofthese pests is the brown

plant hopper, an insect thatcauses severe damage torice plants as it feeds. It alsotransmits damaging patho-

gens such as the grassy-stunt and rag-ged-stunt viruses. In an early effort atengineering resistance to multiple threats,we are collaborating with colleagues inChina and England to incorporate re-sistance to both bacterial blight and thebrown plant hopper into several impor-tant varieties of rice, using cloned genes,including Xa21. As more and more re-

sistance genes are cloned, the numberof available combinations will increaseexponentially.

Transgenic disease-resistant plantshold great commercial promise. Al-though no farmers are actually growingsuch plants yet, U.S. companies are lead-ing the commercialization of othertransgenic crops. The Flavr Savr toma-to, developed by Calgene for increasedshelf life, was the first commerciallyavailable genetically engineered food.Soybeans resistant to the herbicide

Roundup came on the market in 1996;

Making Rice Disease-Resistant104 Scientific American November 1997

RICE LEAVES exposed toXoo resist infection when the geneXa21

is present, both in our transgenic Taipei variety (pair on left) and intraditionally bred plants (pair on right). Infected leaves from sus-ceptible plants develop extensive, yellow lesions (center pairs).

JEFFHALLUniversityofCaliforniaatDavis

JAPONICA SUBSPECIES INDICA SUBSPECIES

ENGINEERED

TAIPEI 309 LINE

CONTAINING

Xa21

SUSCEPTIBLE

TAIPEI 309

LINE

SUSCEPTIBLE

INDICA

LINE

TRADITIONALLY

BRED INDICA LINE

CONTAINING

Xa21



6/6

maize engineered for resistance to her-bicide was recently approved for sale inthe U.S. and Canada.

Industrial nations will probably ben-efit most from the currently availabletransgenic products. In the developingworld, for instance, farmers often can-

not afford technologies that require in-put of expensive herbicides. In contrast,both developing and industrial coun-tries are likely to find disease-resistanttransgenic grains useful. These grainsmay also enjoy more acceptance thancertain other new transgenics (such ascontroversial insecticide-producingplants that some fear will lead quicklyto insecticide-resistant insects). Trans-genic disease-resistant plants may even-tually have a significant effect on the

economics of crop growth, promotingmore efficient land use, better globalfood supply and environmentally safermethods of disease and pest control.

With the commercial promise oftransgenic disease-resistant crops comesa social responsibility. In 1996 Davis

established the Germplasm ResourceRecognition Fund to acknowledge thecontributions of developing nations tothe success of the universitys programs,including Xa21, for which the universi-ty has filed a patent application. Fi-nanced by income from commercializa-tion of genetic materials obtained in theThird World, the fund will provide fel-lowship assistance to researchers fromdeveloping nations. Farmers in thesepoor regions will also be able to obtain

seeds of our transgenic lines at the samecost as the traditional parent lines. Thefund provides a means for University ofCalifornia scientists to patent their in-ventions and make them into commer-cially viable products while recognizingand fostering contributions from the

developing world.The potential of genetic engineering

in rice and other grains will not be ex-hausted with disease resistance. The fu-ture will undoubtedly bring the cloningof many more genes responsible for oth-er valuable traits (cold tolerance, per-haps, or drought resistance). Ultimate-ly, breeders and farmers will be able tochoose from a whole toolboxful ofcloned genesgenes that will let themreap more of what they sow.

Making Rice Disease-Resistant Scientific American November 1997 105

XA21 PROTEINS appear to consist of three parts: a section that detects signalsfrom bacteria (blue), a section that spans the membrane of the rice cell (purple) anda section that generates a message inside the rice cell (orange).

The Author

PAMELA C. RONALD received a Ph.D. fromthe University of California, Berkeley, in 1990.Her graduate work in the laboratory of B. J.Staskawicz focused on the genetic basis of diseaseresistance in tomatoes and peppers. She began herwork on rice disease resistance as a postdoctoralfellow in Steven D. Tanksleys laboratory at Cor-nell University. This work continues at U.C.-Davis,where Ronald is an associate professor. When notworking on the problems of the worlds crops, sheenjoys the harvest from her husbands organic farm.

Further Reading

Plant Diseases: Their Biology and Social Impact. G. Schumann. American Phy-topathological Society, 1991.

Rice, the Essential Harvest. Peter T. White. National Geographic, Vol. 185, No.5, pages 4879; May 1994.

Molecular Genetics of Plant Disease Resistance. B. J. Staskawicz et al. in Sci-ence, Vol. 268, pages 661667; May 5, 1995.

A Receptor Kinase-like Protein Encoded by the Rice Disease ResistanceGene, Xa21. Wen-Yuan Song et al. in Science, Vol. 270, pages 18041806; Decem-ber 15, 1995.

Signaling in Plant-Microbe Interactions. B. Baker et al. in Science, Vol. 276,pages 726733; May 2, 1997.

Sounding the Alarm

How do our blight-resistant rice plantssense bacterial intruders? We thinkthe protein encoded by Xa21acts as a kind ofreceiver; pairs of the proteins most likelyspan the cell membrane, picking up externalsignals sent by the bacteria and relayingthem inside the cell. A protective responseensues: the alerted cell signals its neighborsto mount a defense and then dies; groups ofdead cells prevent further spread of the in-vader. The antennalike part of the protein out-side the cell resembles proteins from animalsthat recognize and bind to other molecules.We have not yet found the bacterial mole-cule that tips off the rice cells to their ene-mys presence, but my group is looking for it.

The portion of protein inside the rice cell isalso familiar: it appears to be a kinase, a com-mon type of enzyme responsible for prod-

ding cells to action. We think this enzymeswitches on in response to the bacterial sig-nal, trumpeting a message throughout thecell to activate defenses. P.C.R.

MOLECULE

RELEASED BY

BACTERIUM

PAIR OF XA21

PROTEINS

DEFENSE

ACTIVATION

AND CELL

DEATH

SIGNALS

RICE PLANT

CELL INTERIOR

CELL

MEMBRANE

J

ENNIFERC.

CHRISTIANSEN

SA


6. arroz resistente

Documents