Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica)

To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.     

Influence of jasmonic acid as potential activator of induced resistance against Karnal bunt in developing spikes of wheat

Induction of defense response against Karnal bunt (KB) by suppressing the pathogenesis was observed upon exogenous application of jasmonic acid (JA) as evident from decrease in the coefficient of infection and overall response value in both susceptible and resistant varieties of wheat. The ultra-structural changes during disease progression showed the signs of programmed cell death (PCD). However, JA strengthened the defense barrier by enhancing the lignifications of cell walls as observed in spikes of both varieties by histochemical analysis. Compared to the plants inoculated with pathogen alone, plants of resistant line (RJP) first treated with JA followed by inoculation with pathogen showed more lignifications and extracellular deposition of other metabolites on cells, which is supposed to prevent mycelial invasions. Contrary to this, susceptible (SJP) lines also showed lignifications but the invasion was more compared to resistant line. Induction of protease activity was higher in resistant variety than its corresponding susceptible variety. The protease activity induced during the colonization of the pathogen and its proliferation inside the host system gets inhibited by JA treatment as demonstrated by the quantitative and in-gel protease assay. The results indicate the role of JA signalling in inhibiting the proteases due to expression of certain protease inhibitor genes. SDS-PAGE analysis shows differential gene expression through induction and/or suppression of different proteins in wheat spikes of resistant and susceptible varieties under the influence of JA. Thus, exogenously applied JA provides the conditioning effect prior to the challenge of infection and induces defense against KB probably by maintaining a critical balance between proteases and protease inhibitors and/or coordinating induction of different families of new proteins.     

Basal expression of abscisic acid inducing immunity against Karnal bunt of wheat

In order to understand the intricate mechanism of differential immunity against Karnal bunt (KB), basal levels of carotenoids, abscisic acid (ABA), total protease and protease inhibitor were determined in resistant and susceptible genotypes during transition from vegetative to developing stages of wheat spikes. The lower levels of carotenoid precursor of ABA in resistant genotype than in susceptible genotype could be explained by more inter-conversion of carotenoids into ABA pool which was evident from the results of determining ABA by enzyme-linked sorbent assay. The ABA was significantly higher in resistant genotype at all stages than in susceptible genotype, while a sharp increase was observed at S2 stage. The activity of total protease was higher at initial stages of resistant genotype and gradually declined in later stages after anthesis. In contrast to the protease activity, a reverse trend was observed for the levels of cysteine protease inhibitor, suggesting a negative correlation with each other. Level of cysteine protease inhibitor was observed to be three-folds higher at S2 stage in the resistant genotype than in the susceptible genotype. The results provided the clue for the involvement of ABA-dependent pathways in upregulation of cystatin that leads to induction of differential immunity against the KB pathogen.     

Activity profile of defence-related enzymes in rice genotypes (Oryza sativa L.) against rice blast (Magnaporthe oryzae)

The control and infected leaf samples of blast resistant and susceptible rice genotypes were evaluated for activities of defence-related enzymes viz., total phenol content, chitinase, phenylalanine ammonia lyase (PAL), β-glycosidase, antioxidative enzymes, superoxide dismutase, peroxidase and ascorbate peroxidase. The level of total phenol content and the activity profile of chitinase, PAL and β-glycosidase significantly increased in both blast-resistant and susceptible rice genotypes with comparatively higher level induction Tetep, NLR-20104 and Swarnadhan the blast-resistant genotypes. The antioxidative enzymes were comparatively higher in the leaf samples of blast-resistant genotypes recording highest increase in NLR-20104 and KJT-5. The constitutive levels of total phenols and activity of defence-related and antioxidative enzymes in the control leaf samples differed among the genotypes and were even higher in the two blast susceptible genotypes (EK-70 and Chimansal). However, the level of induction as evident from the activity profile differences between control and infected leaf samples suggests higher level of induction was more which is indicative of the induced defence response. The genotype recording maximum induction of defence-related and antioxidative enzymes activity could be useful criteria in screening for blast resistant genotype in rice.     

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

Biochemical responses associated with resistance to bacterial stalk rot caused by Dickeya zeae in maize

Bacterial stalk rot (BSR) of maize caused by Dickeya zeae is an important disease in northwest region of India. In the current study, eighty maize lines were evaluated for resistance against this disease. Of these, 20 were moderately resistant, 25 were moderately susceptible and the rest were highly susceptible to BSR. Six lines from each set were randomly selected. Activities of three antioxidant enzymes, viz. phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO) were analysed from these three sets of maize lines representing different levels of resistance. A trend of elevated activity of PAL, POX and PPO was observed in all the three sets of maize lines. The results showed significantly more activity of these three enzymes in moderately resistant than highly susceptible maize lines. The activity of PAL and PPO peaked after 48 hr and of POX after 72 hr of challenge inoculation by D. zeae in all the maize lines. The activity of these enzymes further correlated negatively with disease development. Our results show that PAL, POX and PPO play an important role in contributing towards resistance in maize against BSR.     

Antioxidative enzymes and the Russian wheat aphid (Diuraphis noxia) resistance response in wheat (Triticum aestivum)

A crucial function of antioxidative enzymes is to remove excess reactive oxygen species (ROS), which can be toxic to plant cells. The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the activities of antioxidative enzymes was investigated in the resistant (cv. Tugela DN) and the near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). RWA infestation significantly induced the activity of superoxide dismutase, glutathione reductase and ascorbate peroxidase to higher levels in the resistant than in susceptible plants. These findings suggest the involvement of antioxidative enzymes in the RWA-wheat resistance response, which was accompanied by an early oxidative burst. The results are consistent with the role of ROS in the resistance response and the control of their levels to minimise toxic effects.     

马铃薯不同品种感染早疫病菌后防御酶活性变化

通过测定抗性不同的马铃薯品种接种和未接种情况下叶片内防御酶活性,研究马铃薯品种对早疫病的抗性机制。结果表明,在接种处理后,各品种植株体内苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、多酚氧化酶（PPO）和过氧化氢酶（CAT）活性均提高,抗病品种酶活性增幅高于感病品种,说明上述4种酶与品种抗性有一定的关联。     

Histo-chemical changes induced by PGPR during induction of resistance in pearl millet against downy mildew disease

Plant growth-promoting rhizobacteria Bacillus pumilus strain INR-7 effectively induced downy mildew resistance in pearl millet. The histo-chemical analysis of B. pumilus INR-7 mediated systemic resistance showed that induced resistance is associated with the expression of hypersensitive response (HR), enhanced lignification, callose deposition, and hydrogen peroxide in addition to the increased expression of the defense enzymes β-1,3-glucanase, chitinase, phenylalanine ammonia lyase (PAL), peroxidase (POX), and polyphenol oxidase (PPO). There was rapid expression of HR in the resistant pearl millet as well as the susceptible seedlings induced by treatment with INR-7 after pathogen infection when compared to the susceptible seedlings, which expressed HR at later hours. Examination of inoculated pearl millet tissues by microscopy showed that lignin, callose, and hydrogen peroxide accumulated earlier and to higher levels in resistant and induced resistant seedlings. Accumulation of various defense enzymes was an immediate response to Sclerospora graminicola infection and preceded the development of induced resistance elicited by strain INR-7. Tissue print analysis showed that defense enzymes were found to be localized in the vascular bundles and revealed the visual difference in the expression pattern of β-1,3-glucanase, chitinase, PAL, POX, and PPO whose intensity varied among resistant, INR-7 treated, and susceptible pearl millet seedlings. This study clearly demonstrated that the differences between the responses, susceptible, INR-7 treated or resistant pearl millet seedlings recorded differences in the speed, intensity, and pattern of different histo-chemical responses to S. graminicola infection.     

Role of Phenylalanine Ammonia Lyase and Polyphenol Oxidase in Host Resistance to Bacterial Wilt of Tomato

Plants respond to bacterial pathogen attack by activating various defence responses, which are associated with the accumulation of several factors like defence-related enzymes and inhibitors which serve to prevent pathogen infection. The present study focused on the role of the defence-related enzymes phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in imparting resistance to tomato against bacterial wilt pathogen Ralstonia solanacearum . The temporal pattern of induction of these enzymes showed maximum activity at 12 h and 15 h for PAL and PPO, respectively, after the pathogen inoculation (hpi) in resistant cultivars. Twenty different tomato cultivars were analyzed for PAL, PPO and total phenol content following pathogen inoculation. The enzyme activities and total phenol content increased significantly (P < 0.05) in resistant cultivars upon pathogen inoculation. The increase in enzyme activities and total phenol content were not significant in susceptible and highly susceptible cultivars. The role of PAL and PPO in imparting resistance to tomato against bacterial wilt disease is discussed.     

Fractionated extracts of Russian wheat aphid eliciting defense responses in wheat

It is hypothesized that the interaction between aphids and plants follows a gene-for-gene model. The recent appearance of several new Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), biotypes in the United States and the differential response of wheat, Triticum aestivum L., genotypes containing different resistance genes also suggest a gene-for-gene interaction. However, aphid elicitors remain unknown. This study was conducted to identify fractionated Russian wheat aphid extracts capable of eliciting differential responses between resistant and susceptible wheat genotypes. We extracted whole soluble compounds and separated proteins and metabolites from two Russian wheat aphid biotypes (1 and 2), injected these extracts into seedlings of susceptible wheat Gamtoos (dn7) and resistant 94M370 (Dn7), and determined phenotypic and biochemical plant responses. Injections of whole extract or protein extract from both biotypes induced the typical susceptible symptom, leaf rolling, in the susceptible cultivar, but not in the resistant cultivar. Furthermore, multiple injections with protein extract from biotype 2 induced the development of chlorosis, head trapping, and stunting in susceptible wheat. Injection with metabolite, buffer, or chitin, did not produce any susceptible symptoms in either genotype. The protein extract from the two biotypes also induced significantly higher activities of three defense-response enzymes (catalase, peroxidase, and beta-glucanase) in 94M370 than in Gamtoos. These results indicate that a protein elicitor from the Russian wheat aphid is recognized by a plant receptor, and the recognition is mediated by the Dn7-gene product. The increased activities of defense-response enzymes in resistant plants after injection with the protein fraction suggest that defense response genes are induced after recognition of aphid elicitors by the plant.     

Phenylalanine Ammonia Lyase Activity in Pearl Millet Seedlings and its Relation to Downy Mildew Disease Resistance

Phenylalanine ammonia lyase (PAL) activity was studied in differentgenotypes of pearl millet with varying degrees of susceptibilityto downy mildew disease, after inoculating with Pathotype 1of Sclerospora graminicola. In resistant genotypes, the enzymeactivity significantly increased 24 h after fungal inoculationwhile in the susceptible genotypes, the activity decreased.The increase or decrease in enzyme activity was well-correlatedwith the degree of host resistance to the pathogen. A time-courseof change in activity of PAL after inoculation showed a considerabledifference between resistant and susceptible genotypes. Studieson the activity of PAL in different parts of pearl millet seedlingsrevealed that in the resistant genotype, enzyme activity significantlyincreased at 24 h post-inoculation only in the shoot portion,whereas in mesocotyl and root the activity decreased. In susceptibleseedlings, enzyme activity decreased at 24 h post-inoculationin shoot, mesocotyl and root. The activity of PAL was also foundto be pathotype-specific. Histochemical tests for lignin werepositive in infected cells in the resistant genotypes. The roleof PAL in imparting resistance to pearl millet against downymildew disease is discussed. Key words: Sclerospora graminicola, resistance screening, enzyme activity     

蚜虫(Aphis medicaginis Koch)危害胁迫对不同苜蓿品种体内丙二醛含量及防御性酶活性的影响

研究了蚜虫危害胁迫后不同抗蚜性苜蓿品种叶片内丙二醛含量及防御性酶活性的动态变化。结果表明：在蚜虫刺吸诱导的过程中,高感品种的MDA含量始终高于高抗品种,并且高感和高抗品种均保持上升的趋势;高感品种的SOD、POD和PAL活性始终低于高抗品种,其中高感和高抗品种的SOD和POD活性均表现先上升后下降的趋势,而PAL活性上升到高峰后均趋于稳定;CAT活性在高感和高抗品种间表现为交替的上升下降;高抗品种的PPO活性前期低于高感品种,而后期高于高感品种。由此可见,在蚜虫危害胁迫下,高感和高抗品种间MDA、SOD、POD、PAL和PPO活性的变化与苜蓿的抗蚜性密切相关,均可作为苜蓿抗蚜性鉴定的生理指标,而CAT活性变化与苜蓿抗蚜性的联系有待进一步研究。     

Identification and characterization of a novel bacterial β-glucosidase that is highly specific for the β-1,2-glucosidic linkage of sesaminol triglucoside

A gene (PSTG2) coding for a novel β-glucosidase belonging to glycoside hydrolase family 3 was identified in the vicinity of the previously identified β-glucosidase gene [sesaminol triglucoside (STG)-hydrolyzing β-glucosidase, PSTG1] in the genome of Paenibacillus sp. strain KB0549. Compared with PSTG1, recombinant PSTG2 more specifically acted on the β-1,2-glucosidic linkage of the STG molecule to transiently accumulate a larger amount of 6-O-(β-D-glucopyranosyl)-β-D-glucopyranosylsesaminol.     

Reproduction and development of Russian wheat aphid biotype 2 on crested wheatgrass, intermediate wheatgrass, and susceptible and resistant wheat

The Russian wheat aphid, Diuraphis noxia (Kurdjumov), is an economically important pest of small grains. Since its introduction into North America in 2003, Russian wheat aphid Biotype 2 has been found to be virulent to all commercially available winter wheat, Triticum aestivum L., cultivars. Our goal was to examine differences in Russian wheat aphid reproduction and development on a variety of plant hosts to gain information about 1) potential alternate host refuges, 2) selective host pressures on Russian wheat aphid genetic variation, and 3) general population dynamics of Russian wheat aphid Biotype 2. We studied host quality of two wheatgrasses (crested wheatgrass, Agropyron cristatum [L.] Gaertn., and intermediate wheatgrass, Agropyron intermedium [Host] Beauvoir) and two types of winter wheat (T. aestivum, one Biotype 2 susceptible wheat, 'Custer' and one biotype 2 resistant wheat, STARS02RWA2414-11). The susceptible wheat had the highest intrinsic rate of increase, greatest longevity and greatest fecundity of the four host studied. Crested wheatgrass and the resistant wheat showed similar growth rates. Intermediate wheatgrass had the lowest intrinsic rate of increase and lowest fecundity of all tested hosts.     

Root responses to cereal cyst nematode (Heterodera avenae) in hosts with different resistance genes

The development of cereal cyst nematode (CCN; Heterodera avenae ) induced syncytia in the host roots of infected resistant bread wheat ( Triticum aestivum cv. AUS10894), diploid wheat ( Aegilops tauschii ), barley ( Hordeum vulgare cv. Chebec and cv. Galleon) and in the susceptible wheat cv. Meering and barley cv. Clipper were studied over a period of 13 d. The resistance to CCN in these cereal plants is conferred by the resistance genes Cre1 in the wheat cv. AUS10894, Cre3 in A. tauschii , Ha2 in barley cv. Chebec and Ha4 in barley cv. Galleon. Anatomical observations were made on the development of the syncytia in CCN-infected wheat and barley roots, which carry each of these four sources of resistance genes. Accelerated development of the syncytia in resistant plants, especially in the barley cultivars, was observed. The sites of syncytia development in susceptible wheat and barley were also closely associated with the vascular tissues in the stele, but less so in the resistant plants. The syncytia in the infected susceptible wheat and barley were also metabolically active at day 13. By contrast, the syncytia of resistant wheat plants carrying the Cre1 or Cre3 genes remained extensively vacuolated and less metabolically active. In barley plants with the Ha2 or Ha4 genes, the syncytia appeared non-functional and in early stages of degeneration by day 13 after inoculation.     

Effects of herbivore stress by Aphis medicaginis Koch on the Malondialdehyde contents and the activities of protective enzymes in different alfalfa varieties

The study focused on the dynamics of Malondialdehyde (MDA) contents and the activities of protective enzymes in the leaves of alfalfa varieties with various resistances to Aphis medicaginis Koch. The results showed that susceptible varieties always had higher MDA contents than resistant varieties, and the MDA contents tended to rise in both susceptible and resistant varieties in period of the varieties were pierced and sucked by aphids. Superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activities in susceptible varieties were lower than those in resistant varieties, and in both susceptible and resistant varieties the SOD and POD activities tended to rise at first and then decline, and the PAL activities rose to their peaks and then tended to remain stable. In the susceptible and resistant varieties the catalase (CAT) activities appeared to rise and decline alternatively; the PPO activities in resistant varieties were lower than those in susceptible varieties in early growth, but higher than those in susceptible varieties in later growth. It follows that infested by aphids, susceptible and resistant varieties had the MDA contents, variations of SOD, POD, PAL and PPO activities were closely correlated with their aphid resistances, hence these indexes could be used as physiological indexes for testing aphid resistance of alfalfa, whereas the relations of their CAT activities to their resistances needed to be further studied.