Identification of unique or elevated levels of kernel proteins in aflatoxin-resistant maize genotypes through proteome analysis

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize (Zea mays L.). Resistant maize genotypes have been identified, but the incorporation of resistance into commercial lines has been slow due to the lack of selectable markers. Here we report the identification of potential markers in resistant maize lines using a proteomics approach. Kernel embryo proteins from each of two resistant genotypes have been compared with those from a composite of five susceptible genotypes using large format two-dimensional gel electrophoresis. Through these comparisons, both quantitative and qualitative differences have been identified. Protein spots have been sequenced, and based on peptide sequence homology analysis, are categorized as follows: storage proteins (globulin 1 and globulin 2), late embryogenesis abundant (LEA) proteins related to drought or desiccation (LEA3 and LEA14), water- or osmo-stress related proteins (WSI18 and aldose reductase), and heat-stress related proteins (HSP16.9). Aldose reductase activity measured in resistant and susceptible genotypes before and after infection suggests the importance of constitutive levels of this enzyme to resistance. Results of this study point to a correlation between host resistance and stress tolerance. The putative function of each identified protein is discussed.     

Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production

ABSTRACT Aflatoxins are carcinogens produced by Aspergillus flavus and A. parasiticus during infection of susceptible crops such as maize. Several aflatoxin-resistant maize genotypes have been identified and kernel proteins have been suggested to play an important role in resistance. In the present study, one protein (#717), which was expressed fivefold higher in three resistant lines compared with three susceptible ones, was identified using proteomics. This protein was sequenced and identified as a pathogenesis-related protein (PR-10) based on its sequence homology. To assess the involvement of this PR-10 protein (ZmPR-10) in host resistance of maize against fungal infection and aflatoxin production, the corresponding cDNA (pr-10) was cloned. It encodes a protein of 160 amino acids with a predicted molecular mass of 16.9 kDa and an iso-electric point of 5.38. The expression of pr-10 during kernel development increased fivefold between 7 and 22 days after pollination, and was induced upon A. flavus infection in the resistant but not in the susceptible genotype. The ZmPR-10 overexpressed in Escherichia coli exhibited a ribonucleolytic and antifungal activities. Leaf extracts of transgenic tobacco plants expressing maize pr-10 also demonstrated RNase activity and inhibited the growth of A. flavus. This evidence suggests that ZmPR-10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus.     

Identification of a maize kernel stress-related protein and its effect on aflatoxin accumulation

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize. Through proteomic comparisons of maize kernel embryo proteins of resistant and susceptible genotypes, several protein spots previously were found to be unique or upregulated in resistant embryos. In the present study, one of these protein spots was sequenced and identified as glyoxalase I (GLX-I; EC 4.4.1.5). The full-length cDNA of the glyoxalase I gene (glx-I) was cloned. GLX-I constitutive activity was found to be significantly higher in the resistant maize lines compared with susceptible ones. After kernel infection by A. flavus, GLX-I activity remained lower in susceptible genotypes than in resistant genotypes. However, fungal infection significantly increased methylglyoxal (MG) levels in two of three susceptible genotypes. Further, MG was found to induce aflatoxin production in A. flavus culture at a concentration as low as 5.0 muM. The mode of action of MG may be to stimulate the expression of aflR, an aflatoxin biosynthesis regulatory gene, which was found to be significantly upregulated in the presence of 5 to 20 muM MG. These data suggest that GLX-I may play an important role in controlling MG levels inside kernels, thereby contributing to the lower levels of aflatoxins found in resistant maize genotypes.     

Germination induces accumulation of specific proteins and antifungal activities in corn kernels

ABSTRACT This study examined protein induction and accumulation during imbibition and germination of corn kernels, as well as antifungal activities of extracts from germinating kernels against Aspergillus flavus and Fusarium moniliforme. Genotypes studied included GT-MAS:gk and Mp420, which are resistant to A. flavus infection and aflatoxin accumulation, and Pioneer 3154 and Deltapine G-4666, which are susceptible to A. flavus infection and aflatoxin accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved five protein bands that were present at higher concentrations in germinated kernels than in nongerminated kernels. Western blot analyses revealed that one of these proteins reacted with the 22-kDa zeamatin antiserum, and a zeamatin-like protein accumulated to a higher concentration in germinated kernels. Two protein bands from dry kernels that reacted with ribosome-inactivating protein (RIP) antiserum were identified as the 32-kDa proRIP-like form and an 18-kDa peptide of the two peptides that form active RIP. However, in germinated kernels, two protein bands that reacted with RIP antiserum were identified as two RIP-like peptides with a molecular mass of approximately 18 and 9 kDa. Purified RIP and zeamatin from corn inhibited growth of A. flavus. Bioassays of germinated kernel extracts from all four genotypes exhibited antifungal activity against A. flavus and F. moniliforme, with extracts from the susceptible genotypes showing greater inhibition zones. This study provides evidence of protein induction in corn kernels during imbibition or the early stages of germination, and the induced proteins may be related to our previous findings of germination-associated resistance in the corn kernel, especially in the susceptible kernels.     

Comparison of Kernel Wax from Corn Genotypes Resistant or Susceptible to Aspergillus flavus

ABSTRACT Russin, J. S., Guo, B. Z., Tubajika, K. M., Brown, R. L., Cleveland, T. E., and Widstrom, N. W. 1997. Comparison of kernel wax from corn genotypes resistant or susceptible to Aspergillus flavus. Phytopathology 87: 529-533.Kernels of corn genotype GT-MAS: gk are resistant to Aspergillus flavus. Earlier studies showed that this resistance is due in part to kernel pericarp wax. Experiments were conducted to compare wax from GTMAS: gk kernels with that from kernels of several susceptible commercial hybrids. GT-MAS: gk had more pericarp wax than did the susceptible hybrids. Scanning electron microscopy revealed that GT-MAS: gk kernels appeared rough and showed abundant wax deposits on kernel surfaces. Susceptible kernels appeared much more smooth and lacked the abundant surface deposits observed in GT-MAS: gk. In vitro bioassays showed that kernel wax from GT-MAS: gk reduced A. flavus colony diameter by 35%. Colony diameters on a medium amended with wax from susceptible kernels did not differ from those of controls. Thin-layer chromatography and analyses of chromatograms using NIH Image software showed a distinctive composition for GT-MAS: gk kernel wax. Chromatograms of wax from GT-MAS: gk contained a peak unique to this genotype, but also lacked a peak common to all susceptible hybrids. This is the first report of specific kernel factors involved in resistance to A. flavus in corn.     

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.     

Resistance to Aspergillus flavus in Corn Kernels Is Associated with a 14-kDa Protein

ABSTRACT Corn genotypes resistant or susceptible to Aspergillus flavus were extracted for protein analysis using a pH 2.8 buffer. The profile of protein extracts revealed that a 14-kDa protein is present in relatively high concentration in kernels of seven resistant corn genotypes, but is absent or present only in low concentration in kernels of six susceptible ones. The N-terminal sequence of this 14-kDa protein showed 100% homology to a corn trypsin inhibitor. The 14-kDa protein purified from resistant varieties also demonstrated in vitro inhibition of both trypsin activity and the growth of A. flavus. This is the first demonstration of antifungal activity of a corn 14-kDa trypsin inhibitor protein. The expression of this protein among tested genotypes may be related to their difference in resistance to A. flavus infection and subsequent aflatoxin contamination.     

beta-1,3-Glucanase Activity in Peanut Seed (Arachis hypogaea) is Induced by Inoculation with Aspergillus flavus and Copurifies with a Conglutin-Like Protein

ABSTRACT Infection of peanut (Arachis hypogaea) seed by Aspergillus flavus and A. parasiticus is a serious problem that can result in aflatoxin contamination in the seed. Breeding resistant cultivars would be an effective approach to reduce aflatoxin accumulation. The objective of this study was to investigate the expression of the pathogenesis-related (PR) protein beta-1,3-glucanase and the isoform patterns in peanut seed inoculated with A. flavus. Peanut genotypes GT-YY9 and GT-YY20 (both resistant to A. flavus infection) and Georgia Green and A100 (both susceptible to A. flavus infection) were used in this study. The activities of beta-1,3-glucanase were similar in the uninfected seed of all genotypes, but increased significantly in the resistant genotypes after inoculation in comparison with the susceptible genotypes. An in-gel (native polyacrylamide gel electrophoresis [PAGE]) enzymatic activity assay of beta-1,3-glucanase revealed that there were more protein bands corresponding to beta-1,3-glucanase isoforms in the infected seed of resistant genotypes than in the infected seed of susceptible genotypes. Both acidic and basic beta-1,3-glucanase isoforms were detected in the isoelectric focusing gels. Thin-layer chromatography analysis of the hydrolytic products from the reaction mixtures of the substrate with the total protein extract or individual band of native PAGE revealed the presence of enzymatic hydrolytic oligomer products. The individual bands corresponding to the bands of beta-1,3-glucanase isoforms Glu 1 to 5 were separated on the sodium dodecyl sulfate-PAGE, resulting in two bands of 10 and 13 kDa, respectively. The sequences of fragments of the 13-kDa major protein band showed a high degree of homology to conglutin, a storage protein in peanut seed. Conglutin is reported as a peanut allergen, Ara h2. Our data provide the first evidences for peanut having beta-1,3-glucanase activities and the association with the resistance to A. flavus colonization in peanut seed. We have not directly demonstrated that conglutin has beta-1,3-glucanase activity.     

Structural changes of homogalacturonan,rhamnogalacturonan I and arabinogalactan protein in xylem cell walls of tomato genotypes in reaction to Ralstonia solanacearum

Healthy and Ralstonia solanacearum-inoculated tomato genotypes susceptible or resistant to bacterial wilt including recombinant inbred lines (RILs) deriving from a cross between the resistant genotype Hawaii7996 and the susceptible Wva700 were compared for symptom and bacterial population development, and for the composition and structure of pectic polysaccharides and arabinogalactan proteins (AGPs) of xylem cell walls by immunological staining of tissue prints. Constitutive differences were observed between resistant and susceptible RILs, with a higher degree of methyl-esterification of homogalacturonan (HG) detected by antibody JIM7 in the resistant plants. After inoculation, decreased methyl-esterification of HG indicated by stronger labeling with antibody JIM5 was observed in all susceptible genotypes and in five of eleven resistant genotypes, with a clear increase in the non-blockwise de-esterification pattern of HG (LM7) only in the susceptible lines, indicating the mode of action of the pectinmethylesterase of R. solanacearum. In the susceptible lines infection generally leads to increased branching of rhamnogalacturonan I indicated by the detection of arabinan (LM6) and galactan (LM5) side chains, and of arabinogalactan protein (LM2), while only few of the resistant genotypes reacted with changes in these epitopes. All the resistant, symptomless genotypes contained relatively high pathogen populations in stems. A clear relation between cell wall composition and degree of latent infection of resistant genotypes was not found.     

Proteomic analysis of Colletotrichum kahawae-resistant and susceptible coffee fruit pericarps

Coffee berry disease (CBD) is caused by the fungus Colletotrichum kahawae and is restricted to the African continent, where it generates losses of up to 80 % of coffee production. Weather conditions in certain growing areas at high altitudes in Colombia appear to be very favourable for the development of this disease. Certain genotypes of Coffee arabica are resistant to this pathogen, such as the Timor Hybrid and some Ethiopian accessions. It is important to identify the proteins in these coffee genotypes that are associated with resistance to this fungus. Therefore, we compared the proteomes of two genotypes that are resistant to different isolates of C. kahawae with the proteome of the susceptible coffee genotype Caturra. We optimized the methodology applied for the extraction, cleaning and purification of proteins from the green fruit pericarp at 150 to 170 days after flowering. Through two-dimensional differential gel electrophoresis, proteomic map images were obtained for the resistant and susceptible genotypes. Fifty-two protein spots that were significantly different between the resistant and susceptible genotypes were detected. These protein spots were isolated and sequenced via mass spectrometry. The sequence analysis identified 14 proteins in the Timor Hybrid and 14 in CCC1147 that were associated with resistance and pathogen defence.     

Investigation of in-vitro methods for measuring eggplant resistance to Verticillium dahliae

An assay based on electrolyte leakage (LEA) from callus tissue of Solanum spp. treated with culture filtrates of Verticillium dahliae was useful for screening eggplant genotypes for resistance to Verticillium wilt. Loss of electrolytes from the most resistant genotypes was significantly lower than that from the susceptible genotypes when callus tissue was incubated with a 10% filtrate solution of the virulent isolate 122. No difference was detected with filtrate solutions of the avirulent isolate 809 of the fungus. Among the tested genotypes, the S. melongena lines CCR3 and SM19/14 and the wild species S. torvum were the most resistant in the glasshouse tests and released the smallest amounts of electrolytes. The most susceptible cultivars, Lunga Violetta and Giant of China, produced the greatest leakage of electrolytes. LEA performed on stem segments failed to distinguish between susceptible and resistant genotypes of eggplant.     

Evaluating commercial maize hybrids for resistance to gibberella ear rot

An integral component of breeding maize for resistance to Fusarium graminearum ear rot is the identification of resistant genotypes. Since natural infection is not consistent from year to year, maize researchers must use manual techniques to inoculate the plant material with fungal spores. Information is presented here on site resistance of commercial maize hybrids to F. graminearum over three years and at two locations. Additionally, results of an investigation on the two predominant techniques of inoculating maize, the silk channel and kernel inoculation methods, are reported. Of 61 commercial hybrids tested, only two were ranked as moderately resistant to the fungus by both inoculation methods. These two hybrids also had a stable response to the F. graminearum infection across seven environments when the silk channel inoculation method was used. The majority of the hybrids were ranked as either susceptible or highly susceptible and less than 10% of the hybrids had a stable response to fungal infection. In the investigation of methodology, it was concluded that silk browning would be the least laborious way to identify the ideal time to complete silk channel inoculations. It was found that kernel inoculations using the pin inoculation method should take place between 11 and 15 days after 50% silking to achieve proper hybrid discrimination. Mist irrigation increased mold severity ratings and resulted in greater discrimination between hybrids with varying levels of resistance to F. graminearum infection.     

Tissue-specific components of resistance to Aspergillus ear rot of maize

Aspergillus flavus and other Aspergillus spp. infect maize and produce aflatoxins. An important control measure is the use of resistant maize hybrids. There are several reports of maize lines that are resistant to aflatoxin accumulation but the mechanisms of resistance remain unknown. To gain a better understanding of resistance, we dissected the phenotype into 10 components: 4 pertaining to the response of silk, 4 pertaining to the response of developing kernels, and 2 pertaining to the response of mature kernels to inoculation with A. flavus. In order to challenge different tissues and to evaluate multiple components of resistance, various inoculation methods were used in experiments in vitro and under field conditions on a panel of diverse maize inbred lines over 3 years. As is typical for this trait, significant genotype-environment interactions were found for all the components of resistance studied. There was, however, significant variation in maize germplasm for susceptibility to silk and kernel colonization by A. flavus as measured in field assays. Resistance to silk colonization has not previously been reported. A significant correlation of resistance to aflatoxin accumulation with flowering time and kernel composition traits (fiber, ash, carbohydrate, and seed weight) was detected. In addition, correlation analyses with data available in the literature indicated that lines that flower later in the season tend to be more resistant. We were not able to demonstrate that components identified in vitro were associated with reduced aflatoxin accumulation in the field.     

Influence of competition and host plant resistance on selection in Phytophthora infestans populations in Michigan, USA and in Northern Ireland

Competition between genotypes of Phytophthora infestans was studied by inoculating potato cultivars with differing susceptibility to late blight in field experiments over three years in Northern Ireland, UK, and Michigan, USA. Multiple isolates of six genotype groups of P. infestans were chosen from the local populations in both N. Ireland and Michigan for inoculation of separate field trials planted in 2003, 2004 and 2005. Four cultivars were used in each trial; two (susceptible cv. Atlantic and the partially resistant cv. Stirling) were common to both locations, whereas the two additional cultivars (with partial resistance to late blight) were cvs Santé and Milagro in N. Ireland and cvs Pike and Jacqueline Lee in Michigan. Single-lesion isolates of P. infestans were obtained from leaves at 1% level of infection, characterized using pre-assigned markers and re-assigned to their respective genotype groups. Extreme selection occurred within the population of genotypes of P. infestans in N. Ireland in each year, with different genotype groups dominating the infection of different cultivars. Selection was observed on all cultivars tested, but was greatest on the more resistant cultivars. Over the 3 years, all of the 114 isolates obtained from cv. Milagro belonged to a single group, whereas among the 118 isolates from cv. Atlantic all six groups were represented. By contrast, in Michigan, the US-8 genotype dominated infection in all cultivars in each year; only 12 of 374 isolates characterized belonged to other genotypes (11 US-14 and a single US-10 isolate).     

Screening Luffa germplasm and advanced breeding lines for resistance to Tomato leaf curl New Delhi virus

The two cultivated Luffa species can be severely infected by Tomato leaf curl New Delhi virus (ToLCNDV) with up to 100% yield loss. Here, 52 Luffa genotypes were screened for ToLCNDV resistance after natural field infection. Mean vulnerability index (VI) ranged from 0.00 to 75.33; genotypes IIHR-137 and IIHR-138 had no symptoms (VI 0), 16 genotypes were resistant (VI 0–25), 15 were moderately resistant (VI 26–50), and 19 were moderate to susceptible (VI?>?50). Ten of the most resistant genotypes and five susceptible checks were then challenge-inoculated using whiteflies or sap in an insect-proof net house; only IIHR-137 [L. cylindrica (L.) Roem.] was symptomless (VI 0.00), and 3–5% of plants of IIHR-138 [L. cylindrica (L.) Roem.] and IIHR-Sel-1 [L. acutangula (L.) Roxb.] had only mild symptoms; genotype Arka Prasan was most susceptible (VI 80.96). Asymptomatic plants were confirmed as infected using polymerase chain reaction. Susceptible genotypes rapidly developed leaf curling, then a severe mosaic 10 days post-inoculation. The resistant inbred lines identified are good candidates for a breeding program for ToLCNDV-resistant cultivars.

     

Influence of host diversity on development of epidemics: an evaluation and elaboration of mixture theory

ABSTRACT A spatiotemporal/integro-difference equation model was developed and utilized to study the progress of epidemics in spatially heterogeneous mixtures of susceptible and resistant host plants. The effects of different scales and patterns of host genotypes on the development of focal and general epidemics were investigated using potato late blight as a case study. Two different radial Laplace kernels and a two-dimensional Gaussian kernel were used for modeling the dispersal of spores. An analytical expression for the apparent infection rate, r, in general epidemics was tested by comparison with dynamic simulations. A genotype connectivity parameter, q, was introduced into the formula for r. This parameter quantifies the probability of pathogen inoculum produced on a certain host genotype unit reaching the same or another unit of the same genotype. The analytical expression for the apparent infection rate provided accurate predictions of realized r in the simulations of general epidemics. The relationship between r and the radial velocity of focus expansion, c, in focal epidemics, was linear in accordance with theory for homogeneous genotype mixtures. The findings suggest that genotype mixtures that are effective in reducing general epidemics of Phytophthora infestans will likewise curtail focal epidemics and vice versa.     

Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL180

In plant–pathogen interactions, strong structural and biochemical barriers may induce a cascade of reactions in planta, leading to host resistance. The kinetic speed and amplitudes of these defence mechanisms may discriminate resistance from susceptibility to necrotrophic fungi. The infection processes of two Ascochyta lentis isolates (FT13037 and F13082) on the recently identified ascochyta blight (AB)‐resistant Lens orientalis genotype ILWL180 and two cultivated genotypes, ILL7537 (resistant) and ILL6002 (susceptible), were assessed. Using histopathological methods, significant differences in early behaviour of the isolates and the subsequent differential defence responses of the hosts were revealed. Irrespective of virulence, both isolates had significantly lower germination, shorter germ tubes and delayed appressorium formation on the resistant genotypes (ILWL180 and ILL7537) compared to the susceptible genotype (ILL6002); furthermore, these were more pronounced on genotype ILWL180 than on genotype ILL7537. Subsequently, host perception of pathogen entry led to the faster accumulation and notably higher amounts of reactive oxygen species and phenolic compounds at the penetration sites of the resistance genotypes ILWL180 and ILL7537. In contrast, genotype ILL6002 responded slowly to the A. lentis infection and reaffirmed previous gross disease symptomology reports as highly susceptible. Interestingly, quantification of H₂O₂ was markedly higher in ILWL180 particularly at 12 h post‐inoculation compared to ILL7537, potentially indicative of its superior resistance capability. Faster recognition of A. lentis is likely to be a major contribution to the superior resistance observed in genotype ILWL180 to the highly aggressive isolates of A. lentis assessed.     

玉米抗南方锈病种质标记基因型鉴定与遗传多样性分析

为阐明玉米抗南方锈病种质的标记基因型和遗传背景,利用7个与玉米抗南方锈病3个基因连锁的SSR标记鉴定了38份抗病玉米种质的标记基因型,并采用40个多态性SSR标记对39份抗南方锈病的玉米自交系和6个标准测验种进行了遗传多样性研究。结果表明,7个与抗病基因连锁的SSR标记将38份抗病种质鉴定为17种标记基因型,表明可能存在多样的抗性基因组合方式;辽2204等9份种质仅扩增出齐319的标记基因型,沈136和W456仅扩增出W2D的标记基因型;种质LO932未扩增出与齐319、P25和W2D相同的标记,可能携带新的抗南方锈病基因;相近遗传背景的抗性种质分属不同的标记基因型,表明抗病种质可能携带的抗南方锈病基因在育种选择中发生了分离。40对多态性SSR引物在45份自交系中共检测出115个等位基因变异,平均每对引物检测到2.88个等位基因,变异范围为2~4;平均多态性信息含量为0.4649,变化范围为0.1258~0.6951;通过UPGMA聚类分析,39份抗病材料被划分到以标准测验种为代表的6个杂种优势亚群中,与系谱分析基本一致,这为在育种中合理利用抗源提供了信息。