Ramachandra K. Kini

Infection induced oxidative cross-linking of hydroxyproline-rich glycoproteins (HRGPs) is associated with restriction of Colletotrichum sublineolum in sorghum

Abstract Hydroxyproline-rich glycoproteins (HRGPs) accumulation and oxidative cross-linking is one of the earliest defense responses in plants against pathogen infection. In the present study HRGP accumulation in three sorghum genotypes i.e. SC146 (resistant), cv. SC326 (intermediately resistant) and BTx623 (susceptible) as a response to Colletotrichum sublineolum isolate CP2126 infection is elucidated. HRGPs were monitored by hydroxyproline (Hyp) estimation. In genotypes SC146 and genotypes SC326 there was a significantly higher amounts of Hyp at 2 days after inoculation (dai) compared to genotype BTx623, indicating an infection induced accumulation of HRGPs. Western blot analysis of acid-ethanol extracted proteins with polyclonal antibody raised against pearl millet purified HRGPs identified four bands with molecular masses of ~65, 45, 17 and 14 kDa as HRGPs. Insolubilization of the 45 kDa protein in genotypes SC146 and SC326 upon infection with C. sublineolum indicates a role of this protein in cell wall cross-linking, coinciding with heavier accumulation of hydrogen peroxide. In addition, tissue print analysis using polyclonal antibody of pearl millet HRGPs recognized these cross-linked proteins to be HRGPs. These findings indicated that HRGPs in sorghum is a component of defense reaction against C. sublineolum infection.

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases

Summary We undertook production and inhibition studies of recombinant millet PGIP. Using computational mutagenesis, the most significant binding contact involved in pearl millet PGIP–AnPGII interaction was identified.

Association between accumulation of allene oxide synthase activity and development of resistance against downy mildew disease of pearl millet

AbstractThe present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars.

Serodiagnosis of pearl millet resistance to downy mildew by quantitating cell wall P/HRGP using polyclonal antiserum Pab-P/HRGP

Proline/hydroxyproline-rich glycoprotein (P/HRGP) level in pearl millet genotypes resistant to downy mildew increase after inoculation with the oomycete pathogen Sclerospora graminicola. Using purified P/HRGPs from pearl millet cell walls, polyclonal antibodies (Pab-P/HRGP) were raised in rabbit. Based on this antiserum, an enzyme immunoassay was developed that displays a linearity detection range from 0.01 to 10 μg P/HRGP. Western blot analysis, confirming the induction of three marker P/HRGPs in the infected resistant genotype, and immunocytochemical studies on P/HRGP localization either in epidermal peelings or in suspension-cultured cells demonstrated the specificity of the antiserum. Besides its characterization, Pab-P/HRGP was employed to screen various genotypes of pearl millet for fast, sensitive and specific detection of induced P/HRGPs upon infections. The results presented are discussed with presumed importance to downy mildew disease and the use of this new antiserum in pearl millet screening for disease resistance.

Genetic variation in Fusarium oxysporum f.sp. cubense isolates based on random amplified polymorphic DNA and intergenic spacer

Abstract Fusarium oxysporum f.sp. cubense (Foc) isolates were obtained from research centers and from the hot spot of fusarium wilt ‘Nanjangud’. The morphological features like colony appearance, growth rate, pathogenicity, optimum growth temperature and vegetative compatibility grouping (VCG) were determined. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA (IGS). All the F. oxysporum f.sp. cubense isolates were pathogenic to banana cultivar ‘Nanjangud Rasabale’ but they did not induce any disease symptoms on cultivar ‘Cavendish’. The F. oxysporum isolate did not induce wilt symptoms on either ‘Nanjangud’ or ‘Cavendish’ cultivar. Based on RAPD data, 140 scoreable markers were obtained from 36 random decamer primers. Cluster analysis with Unweighted Pair-Group Method with Arithmetic mean (UPGMA) using genetic distance showed that the isolates belonged to three main groups. In general, isolate 1, 2, 4, 5 and 7 were in one group and isolate 3 and 6 were in another group. The grouping based on IGS data correlated with the grouping of isolates based on RAPD.

Efficiency of RAPD, ISSR and ITS markers in detecting genetic variability among Salacia species sampled from the Western Ghats of Karnataka

Diversity and phylogenetic relationship between four closely related Salacia species, i.e., Salacia chinensis, Salacia macrosperma, Salacia fruticosa and Salacia oblonga, collected from the Western Ghats of Karnataka, India, was assessed. Ten each of RAPD and ISSR primers generated a total of 76 and 68 loci, generating polymorphisms of 92.21 and 89.71%, respectively. Maximum likelihood analysis of the ITS sequences revealed three clades. Dendrogram analyses of RAPD and ISSR revealed two and four clusters, respectively. Overall polymorphism revealed by RAPD was 41.45 ± 10%, ISSR was 33.58 ± 6.52%, and ITS was 25.50 ± 17.25%. Molecular variance revealed significant variance within and among the Salacia species. Tajima’s D neutrality test and Fu’s Fs were negative for all four species, implying presences of rare alleles and population expansion. Comparative study of RAPD, ISSR and ITS for Salacia species has given an insight into the efficiency of each technique in detecting diversity within and among the population sampled in the Western Ghats of Karnataka.