Rice is regarded as one of the most utilised basic nutrient for a major portion of the universe 's population, particularly in Asia, Latin America, the Middle East, and the West Indies. Around 90 per centum of entire rice is produced in Asia entirely ( Mohanty, 2009 ) . Other so Asia, Rice is besides going an of import cereal nutrient and supports lives of many people in other continents like Central America, Europe and Africa. About 50 million dozenss of unsmooth rice demands to be increased per twelvemonth by 2015 to run into the universe 's demand, and the jutting demand for Asia is an extra 38 million dozenss ( Pandey, 2008 ; Mohanty, 2009 ) . Rice is the most of import staple cereal harvest for more than two billion people in Asia ( Mew et al. , 2004 ) and his...
tories for more than 40 per centum of the Calorie ingestion of most Asians ( Pandey, 2008 ) Interestingly, India is the universe 's 2nd largest manufacturer of white rice accounting for approximately 80 % of all universe rice production. Rice in India is the most dominant harvest, and a basic nutrient for the people of the eastern and southern parts of India. The earliest remains of cultivated rice was found in the North and west parts of India and day of the month from around 2000 BC. In India, Rice production is an of import portion of the national economic system ( Library of Congress, 1995 ) .
With the increasing population, demands for nutrient have been increasing. Increasing the productiveness of nutrient by diminishing the badness of biotic and abiotic factor
will be a ambitious undertaking. Plant-parasitic roundworms are the concealed enemy of harvests and are one of the many groups of harmful beings which depend on workss for their endurance and reproduction ( Khan, 2008 ) . Plant-parasitic roundworms can do important harm to about all sorts of harvests but due to their subterraneous wont and microscopic size they remain unseeable to the bare oculus. The estimated one-year output losingss due to plant-parasitic roundworms in the universe 's major harvests are recorded at approximately 12.3 % and 14 % in the development states ( Sasser and Freckman, 1987 ) . In India, recent surveies show that works parasitic roundworms are responsible for both the quantitatively and qualitative output losingss amounting about Rs.240 billion every twelvemonth ( Khan, 2008 ) . Besides this direct harm, these roundworms besides serve as predisposing agents in development of disease composites in association with other microbic agents including Fungi, bacteriums and viruses. Root-knot roundworms, such as Meloidogyne graminicola are known to do significant lessening in rice production. M. graminicola has increasing importance on lowland rice farming systems where H2O preservation consequences in intermittent implosion therapy of Fieldss. Output losingss in rice caused by Meloidogyne graminicola scope from 20-80 % in highland and about 11-73 % in intermittently flooded conditions ( De Waele & A ; Elsen, 2007 ) . Tolerance to plant-parasitic roundworms is besides dependent on H2O direction patterns. Rice assortments demoing opposition to roundworms may stand for the practical and effectual agencies of pull offing these roundworms in little graduated table rice farming systems. The absence of classical works genteelness solutions and the restrictions of chemical intervention besides
represent an first-class chance for biotechnological applications. Because of the deficiency in cognition about the roundworm and works interaction, the roundworm jobs are non recognized decently. More cognition on the interaction between rice and roundworms is of import and necessary to acquire the roundworm job under control.
The most evolutionary advanced versions for works parasitism by works parasitic roundworms are the merchandises called parasitism cistrons ( Gao et al. , 2002 ) . The parasitism proteins are secreted by the roundworm and play a direct function in works parasitism. These parasitism proteins secernments largely originate from the guttural secretory organ cells, but secernments from the chemosensory amphids might besides be of import ( Davis et al. , 2004 ) . Many parasitism cistrons have already been reported from plant-parasitic roundworms including Globodera rostochiensis, Meloidogyne incognita, Heterodera schachtii etc. However, surprisingly no parasitism cistrons from Meloidogyne graminicola have been reported so far. With the above background, the purposes and aims of the present survey are
To clone putative works cell wall degrading enzymes from the rice root-knot roundworm Meloidogyne graminicola
To analyse the cloned putative works cell wall degrading enzymes from the rice root-knot roundworm Meloidogyne graminicola by application of assorted molecular tools and techniques and to analyze its look at different life phases of roundworms.
The experimental work will affect the undermentioned stairss:
1 ) Finding out whether these cistrons are truly present in the DNA/RNA of Meloidogyne graminicola by Polymerase Chain Reaction ( PCR )
2 ) Functional analyses by in situ hybridisation to look into their look in the secretory organ cells of the roundworm
3 ) To look into the look of the cistrons in different life phases of
The ability to infect or to parasitize host workss by the rice root-knot roundworm Meloidogyne graminicola indicates that the roundworms release certain works cell wall modifying proteins to interrupt the works cell wall in add-on to the mechanical shearing of works cell wall by the stylet of the roundworm. The parasitism proteins secreted by Meloidogyne graminicola might include works cell wall modifying enzymes likely to be of procaryotic beginning besides proteins that are capable of placing in the host works cell nucleus, suppressers of host defence, proteins that can mime works proteins as reported in other cyst and root-knot roundworms.
2. LITERATURE REVIEW
2.1. THE HOST PLANT: Rice
Rice ( Oryza sativa ) is cultivated in approximately 150 million hour angle of land worldwide ( Bouman et al. , 2006 ) and the major basic nutrient in south-east Asia and besides normally grown in West Africa and South America. Rice feeds about one half of the universe 's population. The mean output of rice is about 3-4 dozenss per hour angle ( Padgham et al. , 2004 ) . Rice is the grain with the second-highest production in the universe next to maize and the most of import grain with regard to calorie consumption and human nutrition thereby supplying more than one fifth of the Calories consumed worldwide ( Smith, 1998 ) . The cultivation of rice is well-suited to states and parts holding low labour costs accompanied with high rainfall. Although the parent species of rice are native to parts of South Asia and some parts of Africa, trade and exportation of rice over centuries have made it commonplace in many civilizations worldwide.
2.2. RICE FARMING
2.2.1. IRRIGATED RICE
Irrigated rice farming systems history for approximately 55 % of the universe country under rice cultivation and history for 75 % of the universe entire production ( Bridge et al. , 2005 ) Generally, land for rice cultivation is prepared while moisture and H2O kept held in the reservoirs. In this system, seeds are normally pre germinated and grown in the wet seed-beds for a period of approximately 2 hebdomads ( UNCTAD, 2010 ) . Whereas in direct seeding, seeds are often pre germinated and can be broadcasted by manus or spread over the H2O by utilizing aero planes as pattern in developed states. In this agriculture system, production scopes from 5 tons/ hour angle in the instance of rainy season while more than 10 tons/ hour angle in dry season, when accommodating modern engineerings ( UNCTAD, 2010 ) .
2.2.2. THE RAINFED LOWLAND RICE
The rainfed lowland rice histories for 17 % of the universe production merely following to the irrigated rice farming system ( UNCTAD, 2010 ) . Bridge et Al. ( 2005 ) reported that the universe rice country planted under rain fed lowland farming system was about 31 % . It grows on compacted dirt and in bunded Fieldss that are able to retain H2O between 25-50 centimetres of medium degree and 0-25 centimetres of low degree. This non-irrigated rice is feed by the rainwater or by a local response armored combat vehicle. The major concern in this type of production system is the hazard of drouth and unexpected inundations ( UNCTAD, 2010 ) .
2.2.3. UPLAND OR DRYLAND RICE
The highland or dryland rice represents about around 13 % of the entire
universe country under rice cultivation ( Bridge et al. , 2005 ) and histories for 4 % per centum of planetary rice production ( UNCTAD, 2010 ) . Prot and Rahman, ( 1994 ) observed that root parasitic roundworms in this ecosystems were extremely diversified. In this production system, land is prepared for seting rice and the rice is dry-seeded. This type of ecosystem is common in Brazil, Madagascar, India and other parts of Southeast Asia. It was reported that Meloidogyne spp. ( M. graminicola M. incognita, M. javanica and M. oryzae ) and Pratylenchus zeae occurred in this environment. However, other research workers reported different species of Heterodera in highland rice farming system, such as H. oryzicola ( Rao & A ; Jayaprakash, 1978 ) , H. elachista ( Ohshima, 1974 ) and H. sacchari ( Coyne & A ; Plowright, 2000 ) .
2.2.4. DEEPWATER OR FLOOD-PRONE RICE
Deepwater or flood-prone rice is common in parts of Southeast Asia including Bangladesh, Thailand, Cambodia, and in West Africa and South America besides ( UNCTAD, 2010 ) . Bridge et Al. ( 2005 ) reported that deepwater rice farming system occurred in the river deltas of South and South-east Asia and it occupied about 3 % of the universe rice country. In this production system, H2O degree is 1 to 5 metres deep and supplied by rivers, lakes, tides etc. Rice production in this system is normally low due to natural catastrophes like drouth, inundation and besides due to the low production potency of the cultivars which are grown with few inputs. Interestingly, this rice farming system supports more than 100 million people in the universe and
most of them populating on little household farms and chiefly in the rural countries ( UNCTAD, 2010 ) .
2.3. DEFENSE RESPONSE OF RICE
Plants are capable to protect themselves against this invasion by bring oning complex defence mechanisms, such as alterations in biochemical, morphological and molecular features including production of antipathogenic compounds, look of defence related cistrons and programmed cell death ( Van Loon et al. , 2006 ) . Dangl and Jones ( 2001 ) described that the development of mechanisms of virulency which aid pathogens overcome basal defence responses in workss is responsible for the development of specific disease opposition. Plants are known to support themselves from pathogen onslaught by triping a multi constituent defence mechanism. In host works defence, the invasion of pathogens is recognized by proteins encoded by works disease opposition cistrons ( R-gene ) that bind specifically to the pathogen-derived avirulence proteins ( Avr-gene ) . Odjakova and Hadjiivanova ( 2001 ) described that R- and Avr-gene mediated acknowledgment as cistron for cistron acknowledgment. Hussey and Janssen ( 2002 ) mentioned opposition cistrons in wild murphy species against several Meloidogyne spp. and many of them had been successfully incorporated into modern murphy cultivars and other economically of import harvests. Bari and Jones ( 2009 ) and Jones and Dangl ( 2006 ) reported a 'zigzag ' theoretical account of the works immune system. In this system, pathogen associated molecular forms ( PAMPs ) are recognized by the host encoded PRRs ( pattern acknowledgment receptors ) which consequences in PTI ( PAMP triggered unsusceptibility ) . Successful pathogens secrete effecters that suppress PTI. Thus, disease is induced by pathogen effecter triggered susceptibleness
( ETS ) . Plants acknowledge a given effecter and trip effector-triggered unsusceptibility ( ETI ) consequences in disease opposition. Plant disease opposition is enhanced and pathogen growing is restricted by the activation of PAMP triggered unsusceptibility ( PTI ) or effector-triggered unsusceptibility ( ETI ) .
Fig.A A zigzag theoretical account illustrates the quantitative end product of the works immune system. Source- . Jones and Dangl, 2006.
Harmonizing to Odjakova & A ; Hadjiivanova ( 2001 ) , a allergic response ( HR ) is induced and different defence cistrons are expressed by the action of those compounds as secondary couriers. Zinov'eva et Al. ( 2004 ) mentioned that after the reaction against plant-parasitic roundworms, two types of cell decease occurred of which the first 1 was cell mortification and the 2nd type was genetically programmed cell decease or programmed cell death. Systemic opposition against new infections to other works parts could be acquired by the works after this primary infection and this phenomenon was described by Durrant and Dong ( 2004 ) as SAR ( systemic acquired opposition ) . Systemic acquired opposition induces defence that confers durable unsusceptibility against a broad scope of micro-organisms. The defence response of rice to M. graminicola infection has non yet been observed and there is limited information of defence response of rice to other roundworms besides.
2.4. NEMATODE PROBLEMS IN RICE
More than 35 genera and 200 species ( Prot et al. , 1994 ) of roundworms have been reported in rice. These roundworms can be a major restraint to the high outputs of rice. In highland rice, Meloidogyne spp. and Pratylenchus spp. are known to do more harm while
in deep H2O rice, really few roundworms have been reported including M. graminicola, Ditylenchus dipsaci, doing the ufra disease. Aphelenchoides besseyi, causes white tip disease in rice and is found in most ecosystems ( Bridge et al. , 1990 ) , while other roundworms are non distributed homogeneously across the ecosystems ( Prot et al. , 1994 ) . The foliar roundworm parasites, Aphelenchoides besseyi and Ditylenchus dipsaci, cause seeable symptoms in the leaf and hence are detected easy. In add-on to Aphelenchoides besseyi, Hirshmanniella spp. , cause major jobs in irrigated rice. However, there have been studies of M. graminicola or other Meloidogyne species overruning deep H2O rice apart from the observations that a species of Meloidogyne, referred to as M. exigua, occurs in the deep H2O rice farming part in Thailand ( Hashioka, 1963 ; Kanjanasoon, 1962 ; Ou, 1972 ) .
2.5. THE ROOT-KNOT NEMATODES: Meloidogyne GOLDI, 1892
The root-knot roundworms are known to be responsible for one million millions of dollars of economic loss worldwide each twelvemonth on over 5,000 host species ( Sasser and Freckman, 1987 ) . Meloidogyne spp. is considered as one among the most economically detrimental genera of plant-parasitic roundworms on horticultural and field harvests. It is distributed worldwide, obligate parasites of the roots of 1000s of works species, including liliopsid and magnoliopsid herbaceous and woody workss. Root-knot roundworm is distributed both in Torrid Zones and temperate parts ( Dhandaydham et al. , 2008 ) . Harmonizing to the description of Eisenback & A ; Triantaphyllou ( 1991 ) , root knot roundworms, Meloidogyne spp. , are sexually dimorphous ; females are ball-shaped and 0.3-0.7 millimeter in diameter with
a slender cervix embedded in root tissue ; their vulva is present near the anus and is subterminal ; organic structure cuticle is thin, annular and milky. Male roundworms are worm-shaped, 1-2 millimeter long and nonparasitic in dirt ; they have robust spiculums but Bursa is absent. As these roundworms are entoparasites, their stylet is short and reasonably sclerotized. Harmonizing to Hunt et Al. ( 2005 ) , the excretory pore of these roundworms is frequently located near the stylet base ; their eggs are deposited in a gelatinlike matrix which is present outside the organic structure ; the juveniles ( J2 ) of these roundworms are worm-shaped, slender and about 450 Aµm long holding a decrepit sclerotized stylet. Harmonizing to Shurtleff and Averre ( 2002 ) , the mean length of the stylet of the root-knot roundworms is 10-20 Aµm but may hold a small fluctuation among J2, males and females. The systematic place of root-knot roundworms as mentioned by De Ley & A ; Blaxter ( 2002 ) is: Phylum: Nematoda ; Class: Chromadorea ; Order: Rhabditida ; Suborder: Tylenchina ; Infraorder: Tylenchomorpha ; Superfamily: Tylenchoidae ; Family: Meloidogynidae ; Genus: Meloidogyne. De Waele & A ; Elsen ( 2007 ) reviewed that 92 nominal Meloidogyne species had been described by 2006. Major species of Meloidogyne include M. graminicola, M. mayaguensis. M. chitwoodi, M. hapla, M. incognita, M. javanica, M. Arenaria, M. exigua and M. falax. Among these, M. graminicola is considered as the most common and of import roundworm for rice root-knot roundworms harmonizing to Hunt et Al. ( 2005 ) ; De Waele and Elsen ( 2007 ) ; Jepson (
1987 ) .
2.6. THE ROOT-KNOT NEMATODE: Meloidogyne graminicola
2.6.1. Meloidogyne graminicola
M. graminicola is a common species of the Torrid Zones and semitropicss where it infects rice and it is a facultative, meiotic parthenogen, with a monoploid chromosome figure of 18. M. graminicola is a sedentary endoparasitic roundworm and foremost reported by Golden and Birchfield in 1965 from grasses in the United States. The rice root-knot roundworm M. graminicola is known to be established in India, China, Nepal, Bangladesh, Laos, Thailand, United States and Vietnam ( Yik et al. , 1977, 1979 ; Kihn, 1982 ; Poudyal, 2005 ) . De Waele and Elsen ( 2007 ) described Meloidogyne graminicola as the most detrimental Meloidogyne species on rice in shallow intermittently flooded land and highland conditions and besides reported that it was non merely distributed in chief rice bring forthing country, specially the South and Southeast Asia, but was besides found to be distributed in Brazil, Colombia, USA and South Africa and is prevailing in rainfed ( highland ) , irrigated ( lowland ) and deepwater rice ecosystem. Meloidogyne graminicola has been reported from rice turning parts of India ( Isreal, Rao & A ; Rao, 1963 ; Roy, 1973 ) , Laos ( Manser, 1968 ) , Thailand ( Buangsuwon et al. , 1971 ) , U.S.A. ( Golden & A ; Birchfield, 1968 ; Yik & A ; Birchfield, 1979 ) , and Bangladesh ( Hoque & A ; Talukdar, 1971 ; Page et al. , 1979 ) . It has been reported chiefly from rice turning in highland conditions and baby's rooms ( Buangsuwon et al. , 1971 ; Israel, Rao & A ;
Rao, 1963 ; Manser, 1968 ; Rao & A ; Israel, 1971, 1972 ) , and besides reported to be absent from the rice harvest grown in afloat Fieldss ( Buangsuwon et al. , 1971 ; Manser, 1968 ) .
2.6.2. SYMPTOMS OF Meloidogyne graminicola ON RICE
The common symptoms due to Meloidogyne graminicola infection on rice include roots with hooked-like saddle sores, immature foliages with deformed and crinkly mark in borders, slow growing, immature workss cholorosis and the to a great extent septic rice mature and flower earlier than the healthy rice. Deep H2O rice assortments can stretch to come above the H2O surface ; nevertheless, upon terrible infection with M. graminicola they are unable to turn and submerge, go forthing spots of unfastened H2O in afloat Fieldss ( Bridge & A ; Page, 1982 ) . Small saddle sores appear on the roots as beaded, clubbed or spindle-shaped, which coalesce upon heavy infection. Characteristic hooked and conceited root tips are evident which prevent root elongation ( De Waele & A ; Elsen, 2007 ) .
Fig. Meloidogyne graminicola root saddle sores on rice seedlings. Source-http: //www.warda.org/publications/Warda_Nemaotde.pdf. 6thDec2010.
Fig.Characterisics hooked tips caused by Meloidogyne graminicola. Source-http: //www.warda.org/publications/Warda_Nemaotde.pdf. 6thDec2010.
2.6.3. LIFE STAGES OF Meloidogyne graminicola
The 2nd phase juveniles or J2 phases of M. graminicola penetrate the root behind the root tip. This leads to giant cell formation due to feeding by juveniles and hypertrophy of cells which farther leads to the development of saddle sores. The saddle sore so continues to swell, with females of J4 and males of J4 inside the saddle sores. As the life rhythm goes on, the male leaves the root while the female corsets inside
the root and starts puting eggs in a gelatinlike matrix outside the root. J2 hatch from the eggs and are attracted towards the root and in this manner, the life rhythm carries on. The life rhythm depending on the environmental conditions takes around one to two months. However, there are studies that the life rhythm of the rice root-knot roundworms under favourable conditions at 25-30A°C takes 19 yearss ( Bridge & A ; Page, 1982 ) . M. graminicola usually provenders on the cells and interferes with alimentary consumption, H2O consumption, and translocation due to root harm ( Caillaud et al. , 2008 ) . Gheysen and Jones ( 2006 ) described that juveniles of Meloidogyne entered the root tip and migrated intercellularly towards the root tip. After making the root vertex, they turned back and migrated once more intercellularly until they found a suited topographic point near vascular cylinder for feeding site initiation. Karsen and Moens ( 2006 ) besides reported that like other root-knot roundworms, M. graminicola started feeding on a group of elephantine cells established in the bast or next parenchyma. Rao and Israel ( 1973 ) reported that this roundworm completes its life rhythm in 26-51 yearss in India on the other manus ; Bridge and Page ( 1982 ) reported that this nematode species completes its life rhythm in less than 3 hebdomads at 22-29A°C in Bangladesh, ensuing in constructing up of immense population densenesss during a individual harvest rhythm.
Fig. Life rhythm of Root knot nematode. Source-
hypertext transfer protocol: //www.apsnet.org/edcenter/intropp/lessons/Nematodes/Pages/RootknotNematode.aspx
2.6.4. RICE YIELD IN RESPONSE TO M. graminicola INFECTION
The rice root-knot roundworm can assail the rice works during all the growing
phases and considered as one of the restricting factors in rice production. There is an estimated decrease of 2.6 per centum in the output of rice for every 1000 roundworms present in immature seedlings as recorded in highland rice farming system and in irrigated rice ( Rice knowledge bank, 2010 ) . Greatest output diminution is reported under non flooded conditions ( Plowright & A ; Bridge, 1990 ; Prot & A ; Matias, 1995 ; Tandingan et al. , 1996 ; Soriano et al. , 2000 ; Soriano & A ; Reversat, 2003 ) nevertheless, great output losingss can besides happen in drought-prone rain fed lowland systems ( Padgham et al. , 2004 ) . Soriano et Al. ( 2000 ) recorded output losingss due to M. graminicola which ranged from 11 % to 73 % in simulations of intermittently flooded rice.
2.6.5. MANAGEMENT PRINCIPLES OF Meloidogyne graminicola
There are assorted control measures that are available for pull offing rice root-knot roundworms. This includes biological, physical, chemical, cultural, immune cultivars and mechanical control. The common cultural controls that are put into patterns include uninterrupted implosion therapy of the rice field, raising the rice seedlings in afloat dirts and besides harvest rotary motion. M. graminicola control can be achieved by uninterrupted implosion therapy of rice workss in deep H2O ( Bridge et al. , 2005 ) . Rao and Israel ( 1971 ) and Soriano et Al. ( 2000 ) proposed that early-season implosion therapy can cut down M. graminicola harm on rice as the Meloidogyne juveniles can non occupy the roots in afloat conditions. Crop rotary motion with marigold ( Tagetes sp. ) is besides found
to be effectual in take downing the root-knot roundworm populations due to its nematicidal belongingss. There are several nematicidal compounds which can be used as chemical control. Other control methods includes the application of chemicals like fensulfothion or oxamyl, carbofuran, phorate etc. Soriano and Reversat ( 2003 ) reported that carbofuran controlled the rice root-knot roundworm and improved output of the first rice harvest but did non impact the 2nd rice harvest.
2.7. NEMATODE PARASITISM GENES
Root-knot roundworms which are extremely successful parasites evolved a really specialised eating relationship with the host works or harvests to do the destructive root-knot disease. They initiate their parasitic relationship with the host by let go ofing their secernments into root cells which in bend stimulate the root cells of the host to go specialised eating cells which are considered as the individual beginning of foods indispensable for the roundworm 's endurance ( Baum et al. , 2007 ) . A deeper apprehension of the basic rules and mechanisms of root-knot nematode parasitism is critical for detecting new marks in root-knot roundworms to develop fresh harvest opposition utilizing biotechnology tools.
Fig. Model of possible interactions of some secreted merchandises of nematode parasitism cistrons with host works cells. Source- Davis et al. , 2004
The parasitism cistrons expressed in the root-knot roundworm 's esophageal secretory organ cells encode secretory proteins that are released through its stylet to direct the interactions of the roundworm with its host workss ( Huang et al. , 2005 ) . The merchandises which are jointly called parasitome, of parasitism cistrons secreted into susceptible host tissue modulate the complex alterations in map, morphology and cistron look in host root cells
to organize feeding cells. Plant-parasitic roundworms are good equipped with a stylet to rupture the cell walls and allow exchange of solute between works and parasite and besides have well-developed secretory secretory organ cells associated with the gorge that produce secernments released through the stylet into host. In order to be parasitic, the roundworm must be able to perforate the roots of host workss and migrate through root tissues. Despite the little size of root-knot and cyst nematode morbific J2 phase, works cell walls serve as an obstructions so nematodes release a mixture of cell-wall-digesting enzymes to interrupt structural works cell-walls ( Baum et al. , 2007 ) Above this, the most interesting things appear to be the nematode-directed formation of the eating cells by both the root-knot roundworms as giant-cells and cyst roundworms as syncytia. Gheysen and Jones ( 2006 ) besides suggested that both initiation and care of elephantine cells were controlled by stylet secernments. These secernments were originated from dorsal and subventral guttural secretory organs of feeding roundworms. They besides reported that the nematode cistrons expressed entirely in subventral secretory organ cells were most similar to the cistrons that produced cell wall debasement enzymes from bacteriums and were non present in symbiotic bacteriums. Zinov'eva et Al. ( 2004 ) summarized several cistron merchandises isolated from subventral secretory organs of roundworms which included lipoprotein, cellulose-binding protein, endoglucanase, chitinase, pectinase and protease.
2.8. PARASITISM GENE IDENTIFICATION
A figure of attacks to place the nematode parasitism cistrons and proteins have been devised and tried. Many of these attacks chiefly focus on esophageal-glands due to their active engagement in parasitism. The designation of parasitism cistrons has proven to
be hard due to the microscopic size of the plant-parasitic roundworms, which makes it difficult to roll up adequate stuff for analysis ( Vanholme et al. , 2004 ) . The peptide sequence from an antigen purified with an esophageal-gland-specific monoclonal antibody was used to insulate the first parasitism cistron from a plant-parasitic roundworm ( Smant et al. , 1998 ) , encoding a I?-1,4-endoglucanase enzyme ( cellulase ) . Immunoaffinity purification was used to enrich secreted proteins, which resulted in the determination of a secreted protein ( endo-1, 4- I?-glucanase ) from the subventral secretory organs of the cyst roundworm Globodera rostochiensis ( Smant et al. , 1998 ) . Another method to be mentioned is the analysis of gathered nematode secernments by 2D gel cataphoresis and microsequencing. This has besides proved successful for the Beta vulgaris cyst roundworm Heterodera schachtii ( De Meutter et al. , 2001 ) and the root-knot roundworm Meloidogyne incognita ( Jaubert et al. , 2002a ) . Recently, mass spectometry has besides been used for direct designation of proteins secreted by M. incognita, uncovering proteins with host cell reprogramming possible ( Bellafiore et al. , 2008 ) . Further surveies besides reveal the look of cyst nematode endoglucanase cistrons and their associated merchandises specifically from subventral secretory organ cells of the roundworm, cellulolytic activity of the enzymes, and secernment of cellulases from the stylet in the works during migration of morbific J2 phase inside the host roots ( Wang et al. , 1999 ) . These surveies besides confirmed and refined the old studies of secreted cell-wall-modifying enzymes from works parasitic roundworms ( Deubert et al. 1971 )
. An of import find of the roundworm endoglucanases was their strong resemblance to prokaryote ( glycosyl hydrolase household 5 ) endoglucanases and small similarity to endoglucanases of eucaryotes and no similarity to any cistron of Caenorhabditis elegans ( Smant et al. , 1998 )
2.9. Beginning OF NEMATODE PARASITISM GENES
Plant-parasitism is believed to hold evolved at least three times independently ( Blaxter et al. , 1998 ) . The cistrons that were evolved from nematode ascendants of modern-day species are one likely possible mechanism for the beginning of nematode parasitism cistrons and the other mechanism may be horizontal cistron transportation ( HGT ) . It was reported that those cistrons expressed in the esophageal secretory organ cells of plant-parasitic roundworms show strongest similarities to the bacterial cistrons which strengthened the bing hypothesis that parasitism cistrons in plant-nematodes may hold been acquired, at least in portion, by horizontal cistron transportation from bacteriums and other micro-organisms that inhabit the same parasitic environment. The cistrons Mj-cm-1and Mi-cbp-1 shows strongest similarities to the cistrons of bacteriums ( Dinging et al. , 1998: Lambert et al. , 1999 ) . The complementation of a bacterial mutation with Mj-cm-1 was besides used to supply functional analysis of the cistron ( Lambert et al. , 1999 ) . Most of the parasitism cistrons are found to be extremely similar to bacterial sequences thereby proposing that these parasitism cistrons could hold been acquired from bacteriums through horizontal cistron transportation ( Danchin et al. , 2010 ) . For illustration, the nematode endo-1,4-I?-glucanases from the Tylenchomorpha, which belong to Glycosyl Hydrolase Family ( GHF5 ) , show less similarity to works endoglucanases but show
resemblance to the bacterium. The cistrons encoding the cellulases enzymes of both roundworms and bacteriums may hold evolved from an ancient cellulase of a common ascendant of both the bacterium and roundworms. The endoglucanases from roundworms show the highest similarity with the bacterial 1s, which besides points to a HGT from bacterium to an ascendant of the cyst roundworm. However, it is non possible and advisable to supply the conclusive grounds for a horizontal cistron transportation ( HGT ) from one being to another being source line. There are illustrations of putative instances of horizontal cistron transportation from eucaryote to prokaryote, prokaryote to prokaryote and from procaryote to eukaryote ( Smith et al. , 1992: Syvanen, 1994 ) . On the other manus, the presence of bacterial symbionts in nematode ascendants, such as the bacteria Wolbachia symbiont found in filarial roundworms ( Blaxter et al. , 1999 ) , may besides stand for a beginning for transportation of familial stuff from bacteriums to roundworms.
2.10. PLANT CELL WALL STRUCTURE AND COMPOSITION
Plant cell walls are complex mixture of saccharides, proteins, lignin, H2O and other substances including cutin, suberin, with inorganic compounds that differ from works to works species and even cell types. This composing and structural fluctuation is further increased due to assorted developmental events and exposure to assorted abiotic and biotic emphasiss ( Showalter, 1993 ) . The major saccharides in the turning works cell wall consist of cellulose, hemicellulose and pectin. The cellulose microfibrils are linked to hemicellulose organizing the web of cellulose-hemicellulose which is embedded in the pectin matrix. Plants cell walls are composed of three types of layers- the in-between gill, the primary
cell wall and in some instance the secondary cell wall. The in-between gill is found to be rich in pectin and deposited after mitosis and connects the two next works cells ( Cosgrove, 2005 ) . The major polyoses in the primary cell wall are cellulose, pectin and hemicellulose whereas secondary cell walls are composed of cellulose, lignin, and hemicellulose like xylan, glucuronoxylan, arabinoxylan and glucomannan. Structural proteins are besides found in most works cell walls and are classified as Hydroxyproline rich glycoproteins ( HRGP ) , Glycine-rich proteins ( GRPs ) , Arabinogalactan proteins ( AGP ) and Proline-rich proteins ( PRPs ) . These proteins are found to be concentrated in specialised cells and in cell corners besides. Plant cell walls of the cuticle and endodermis may besides incorporate suberin or cutin, two polyester-like polymers that protect the cell from herbivores ( Moire et al. , 1999 ) . Plant cells walls besides contain legion enzymes, such as hydrolases, peroxidases, I±-mannosidases, I?-mannosidases, I?-1,3-glucanases, I?-1,4-glucanases, polygalacturonase, saccharases, malate dehydrogenase, arabinosidases, pxylosidases, peptidases and ascorbic acid oxidase ( Varner and Lin, 1989 ) .
2.11. NEMATODE PLANT CELL WALL DEGRADING ENZYMES
The most widely studied cell wall degrading enzyme in roundworms till day of the month is cellulase or endo-1, 4-I?-glucanase. These enzymes degrade the cellulose which is the structural constituent of the works cell wall. The cellulases were identified in the sedentary roundworm genera Heterodera, Globodera and Meloidogyne ( Smant et al. , 1998 ) . It belongs to the glycosyl hydrolase household 5 ( GHF5 ) and are believed to be adopted from bacteriums through horizontal cistron transportation ( HGT ) ( Jones et
al. , 2005 ; Danchin et al. , 2010 ) . The works cell wall digesting enzymes cellulase and pectinase have been described for root-knot roundworms ( Huang et al. , 2004 ; Huang et al. , 2003 ; Huang et al. , 2005 ; Rosso et al. , 1999 ) and cyst roundworm species ( Smant et al. , 1998: Gao et al. , 2003 ; Wang et al. , 2001 ; Yan et al. , 2001 ) . The first major accomplishment in parasitism cistron find was the find of cellulase cistrons from the soya bean and murphy cyst roundworms. The find of cellulase cistrons was really of import since no cellulase cistrons had been reported from animate beings at that clip ( Smant et al. , 1998 ) . The enzymes beta-1, 4-endoglucanase cistrons from Pratylenchus penetrans ( Uehara et al. , 2001 ) , a migratory parasite that besides requires enzymes to come in the works cell-walls was reported subsequently. An enzyme of the beta-1, 3-endoglucanase type was besides late reported from Bursaphelenchus xenophilus, the pinewood roundworm where it is hypothesized of being involved in nematode eating from the fungous mycelium ( Kikuchi et al. , 2005 ) .
2.11.2. PECTATE LYASE
The enzyme pectate lyase can split the internal ( 1, 4 ) -I±-linkages of pectate and was identified in Meloidogyne spp. , but has besides been reported from other genera such as Heterodera, Globodera and Bursaphelenchus ( Popeijus et al. , 2000 ; Doyle & A ; Lambert, 2002 ; Huang et al. , 2005 ; Kikuchi et al. , 2006 ; Vanholme et al. , 2007 ; Abad et al.
, 2008 ) . The pectinase proteins obtained from nematode were of the type pectate lyase which are found in Fungi and bacteriums, cyst and root-knot roundworms ; ( Popeijus et al. , 2000 ; Huang et al. , 2003 ; Huang et al. , 2005 ; De Boer et al. , 2002 ; Doyle & A ; Lambert, 2002 ) or to the polygalacturonase type of bacteriums ( Jaubert et al. , 2002 ) . The engagement of these enzymes in incursion and migration is good supported by the grounds that these enzymes are produced and released during nematode incursion and migration and to a smaller extent, or non at all during the sedentary phases of the roundworms ( Huang et al. , 2005 ; Rosso et al. , 1999 ; De Boer et al. , 1999 ; Goellner et al. , 2000 ) . These types of cell wall digesting enzymes are besides reported from outside the community of sedentary roundworms.
The Mi-pg-1 cistron encoding a functional polygalacturonase ( PG ) from M. incognita was the first known carnal polygalacturonase ( PG ) cloned ( Jaubert et al. , 2002 ) . These enzymes help in catalysing the hydrolysis of pectic polygalacturonic acid and in bend release oligogalacturonides. PGs are classified into two categories viz. exo-PGs and endo-PGs depending on their manner of action. The cistron Mi-pg-1 encodes a 633 amino acid protein. Phylogenetic analysis reveals that Mi-pg-1 is closer to PGs from procaryotes than to eucaryotic enzymes. The close similarity between bacterial PGs and Mi-pg-1 provides strong grounds back uping the hypothesis that the parasitism cistrons in roundworms may hold been acquired
through cistron transportation from micro-organisms. More interestingly, M. incognita PG could play an of import function in weakening the works cell walls of root tissue during nematode incursion and intercellular migration by the parasite like other nematode parasitism cistrons.
The most abundant polyose in nature following to cellulose is xylan and is composed of ( 1, 4 ) -I?-linked xylopyranose units ( Collins et al. , 2005 ) . The enzymes endo-1,4-I?-xylanases depolymerise the nonhydrolysed xylan polymer by spliting the xylan anchor ( Subramaniyan & A ; Prema, 2002 ) . The word picture of the first functional animate being endo-1, 4-I?-xylanase cistron was reported from Meloidogyne incognita, the southern root-knot roundworm. The nematode endoxylanase, Mi-XYL1, has similarity to bacterial endoxylanases ( Mitreva- Dautova et al. , 2006 ) . Most of these enzymes have been designated to the household GHF5, although there is some confusion about this categorization since the proteins have similarity to GHF30 enzymes every bit good. Expressed sequence ticket ( EST ) survey on Radopholus similis, the migratory roundworm revealed some interesting cistrons, including an Eastern time with homology to an endo-1,4-I?-xylanase that was reported late by Jacob et Al. ( 2008 ) and analyzed by Haegeman et Al. ( 2009 ) .
There is grounds that the murphy cyst roundworm besides secretes a protein that has the ability to interrupt the non-covalent bonds in works cell walls in add-on to the ability of interrupting down the covalent bonds found in works cell-walls through the enzyme cellulase and pectinase ( Baum et al, 2007 ) . This type of activity is accomplished by an expansin-like protein found in the murphy cyst
roundworms ( Qin et al. , 2004 ) , which is besides the first confirmed study of such protein from outside the works land. Expansins are involved in softening the works cell-walls by interrupting the non-covalent bonds between cell-wall-fibrils. The attendant cell-wall softening could besides be demonstrated for the murphy cyst roundworm expansin parasitism protein ( Qin et al. , 2004 ) .
2.12. OTHER PARASITISM GENES
2.12.1. CHORISMATE MUTASE
Chorismate is the precursor for a figure of compounds like cellular aromatic amino acids and the works endocrine indole-3-acetic acid, related to salicyclic acid and other secondary metabolites ( Dewick, 1998 ) . This chorismate-derived compounds plays an of import function in works growing and development, in works defence, and besides in interactions with other beings ( Schmid and Amrhein, 1995 ; Weaver and Hermann, 1997 ) . The enzyme chorismate mutase catalysed the pericyclic claisen-like rearrangement of chorismate to prephenate in the shikimate tract, which is a primary metabolic tract found in workss and other microorganisms ( Romero et al, 1995 ) . This enzyme is good characterized from bugs and workss, and non described from any other animate beings outside the plant-parasitic roundworms ( Roberts et al. , 1998 ; Romero et al. , 1995 ; Schmid and Amrhein, 1995 ) . The first carnal chorismate mutase cistron ( Mj-cm-1 ) was cloned from Meloidogyne javanica and found to be expressed in the oesophageal secretory organ cells of the roundworm ( Lambert et al. , 1999 ) . The enzyme chorismate mutase is known to be involved in early development of the eating sites induced by the works parasitic roundworms, but how this enzyme alters the
development of works cells is non decently known ( Doyle and Lambert, 2003 ) . Chorismate mutase was besides identified from soya bean and murphy cyst roundworms ( Bekal et al. , 2003 ; Gao et al. , 2003 ; Jones et al. , 2003 ) .
Chitinase is a putative parasitism protein, identified from the subventral secretory organs of the soybean cyst roundworm ( Gao et al. , 2002 ) . This parasitism protein has a clearly defined map but no clear function for this map during the production of protein. The happening of chitin in roundworm has been found merely in the egg shell ( Bird et al. , 1991 ) and the presence of this parasitism protein chitinases has been discussed as holding a function in the hatching of roundworm ( Baum et al. , 2007 ) . In situ look ( Gao et al. , 2002 ) and microarray look surveies demonstrate that chitinase is non found to be expressed in the eggs of roundworms but has a strong look extremum in the early stages of parasitism after incursion inside the workss ( Baum et al. , 2007 ) .
Annexin cistrons represent a household that codes for Ca dependent phospholipid binding proteins and has a wide scope of reported maps. The messenger RNA for a secretory isoform of an annexin-like protein was besides reported to be expressed in the dorsal secretory organ of the soybean cyst roundworm ( Gao et al. , 2002 ) . However, no clear verification about its function in parasitism can be drawn at this clip. This cistron is besides reported from Globodera pallida, the murphy
cyst roundworm ( Baum et al. , 2007 ) .
Calreticulin-like proteins are besides reported to be secreted from other works parasitic roundworms and are regarded as good campaigners for a function in parasite-host interactions ( Nakhasi et al. , 1998, Pritchard et al. , 1999 ) . A calreticulin-like protein preceded by a signal peptide was reported to be secreted from the subventral secretory organs of a root knot roundworm ( Jaubert et al. , 2002 ) . The confusing array of putative or demonstrated calreticulin maps reported ( Nakhasi et al. , 1998 ) make it hard to corroborate its function in host parasitism by the root-knot roundworms.
2.12.5. SMALL BIOACTIVE PEPTIDES
The most normally expressed parasitism cistron in Heterodera glycines, the soya bean cyst roundworm was foremost identified as ringer HG-SYV46 ( Wang et al. , 2001 ) . The computational analyses found out that the C-terminal sphere of HG-SYV46 is related to the members of the CLAVATA3-ESR-like ( CLE ) household of signaling proteins in Arabidopsis ( Olsen and Skriver, 2003 ) . The CLAVATA3 in Arabidopsis has been identified as a cardinal factor finding shoot meristem distinction ( Fletcher et al. , 1999 ) . The look of the complementary DNA of Heterodera glycines CLAVATA3-like peptide in the clavata3 ( clv3 ) Arabidopsis mutation was found to reconstruct the wild-type phenotype being the first study of ligand apery in works roundworm interactions ( Wang et al. , 2005 ) . It will be interesting to happen out the function of the little C-terminal extension of the cyst roundworm ubiquitin extension proteins when sing the importance of little peptides in signaling functions of
works development and works roundworm interactions ( Tytgat et al. , 2004 ; Gao et al. , 2003 ) . The function of little peptides in nematode-plant interactions is besides confirmed by an unknown peptide fraction smaller than 3 kilo Dalton isolated from potato cyst roundworm secernments that affects works cell division ( Goverse et al. , 1999 ) .
2.13. GENOMES OF M. incognita AND M. hapla
The complete genome sequencing of two root-knot roundworms reveals that M. hapla encodes about 14,200 cistrons in a compact 54 Mbp genome whereas the 86 Mbp of M incognita genome encodes about 19,200 cistrons ( Abad et al. , 2008 ; Bird et al. , 2009 ) . It was reported that M. incognita has a set of 61 works cell wall degrading carbohydrate enzymes but a few of these enzymes were identified antecedently in some plant-parasitic roundworms and in insect ( Caillaud et al. , 2008 ; Davis et al. , 2004 ; Wei et al. , 2006 ) . All together, 21 cellulases and 6 xylanases enzymes from household GHF5, 2 polygalacturonases enzymes from household GHF28 and 30 pectate lyases enzymes from household PL3 were identified from M. incognita ( Abad et al. , 2008 ) . Two extra works cell wall degrading enzymes arabinases ( household GH43 ) and two saccharases ( household GH32 ) were besides identified. Invertase is an enzyme that catalyses the transition of saccharose into glucose and fruit sugar, which the M. incognita utilizations as a C beginning. The enzymes expansins and saccharases were likely acquired through horizontal cistron transportation as most proteins were similar to bacteriums. M. incognita besides secreted four
metabolic enzymes chorismate mutases ( Lambert et al. , 1999 ) , which are extremely similar to bacterial enzymes. Almost half of the cistrons in Meloidogyne hapla show the highest similarities to Caenorhabditis elegans cistrons as based on database similarity and the 2nd largest group demoing similarity is the animal-parasitic roundworms ( Opperman et al. , 2008 ) M. hapla encodes the enzyme cellulases at six venue. Merely four of this has EST support which suggests that two of them are pseudogenes. The most attracting illustration of a root knot nematode cistron acquired through horizontal cistron transportation is pectate lyase. It was originally discovered as a secreted protein ( Davis and Mitchum, 2005 ; Vanholme et al. , 2004 ) . The genome sequence of M. hapla sequence reveals that M. hapla encodes a household of 22 pectate lyases which is common in works infective Fungis ( Opperman et al. , 2008 ) .
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