| Ear rot caused by Fusarium moniliforme (FM) is a destructive disease for its decrease of grain yield in maize, one of the important crops for food in Asia. Especially, a high incidence of ear rot occurs in the moist and warm regions of Southwest China, as well as other regions with similar longitude in other countries. To this end, it is great important to isolate resistant genes with potential function in maize, and to use these genes to breed efficient, broad-spectrum and stable-resistant cultivars to the maize ear rot or understand the defense mechanisms to combat invasion in maize ear rot. In our study, we used two maize cultivars, Bt-1 and Ye478 as completely resistant and highly susceptible to FM due to many years evaluation of field in southwest of China to characterize the specific host response to FM infection. Milky stage maize plants were inoculated with high pathogenic fungus anastomosis groups Fusarium moniliforme on each bract by injection. The inoculated plants were grown under controlled conditions at Maize Research Institute of Sichuan Agricultural University, and bract tissues were collected six times with a 24 h interval after inoculation. To better know the processes involved of defense in maize ear rot upon the FM infection, the time-course infected bracts were observed through Scanning Electron Microscope (SEM) to demonstrate pathogen progression in maize bracts. Then, the biochemical and physiological enzymes activities were analyzed of the both cultivars. In the follow, we identified the differentially expressed genes responding to FM-infection in maize by suppression subtractive hybridization (SSH) and Affymetrix GeneChip Maize Genome Array. The main results are as follows:1. The disease incidence of the both cultivars after FM-infection was investigated in the field. The results showed that the disease incidence was accounted for 1.5%in Bt-1 and mean that this inbred line is belonged to high resistance to FM according to resistant standard. The Ye478 is belonged to high highly susceptible to FM for its 59.4%disease incidence.2. The invasion procedure of fungus on bracts was investigated through SEM. Inoculated and mock-inoculated bract tissues were picked randomly by collecting at 24 h intervals for three independent biological replicates, each consisting of the independent maize bract. FM invasion on the maize bracts of resistant Bt-1 cultivar and the susceptible Ye478 cultivar were observed through SEM at 24 h intervals after inoculation for six stages. During the thin and weak early stage (around 2-3 d), the hyphae of FM developed and expanded on the bracts surfaces of both cultivars. After becoming significantly strong and fine cylindrical, the hyphae gradually moved and infected the maize bract through the stoma at approximately 72 hpi. With the increasing invasion of FM, more and more hyphae assembled as mass into stoma by 120 hpi in both cultivars. However, the invasion of hyphae into stoma was delayed in resistant cultivar Bt-1. This phenomenon reflects more possible defense mechanisms in cultivar Bt-1 were activated than that in Ye478.3. To study the production of enzymes involved in the defense of FM infection, the phenylalanine ammonia-lyases (PAL), peroxidase (POD) and malondialdehyde (MDA) activities were detected during a 6-d time period. In addition, patterns of POD isozymes were also detected to demonstrate POD isozymes for specific different two maize cultivars. PAL and POD were increased promptly higher and quicker in Ye478, comparing to that in resistant cultivar Bt-1. The content of MDA was higher in Bt-1 than Ye478. Finally, the patterns of POD isozyme changed dramatically and increased three or four belts in both cultivars after infection. The result demonstrated that the host might be increase POD activity and isozyme bands to resistant the exogenous pathogen attack for protecting host organism. In a word, our results implies that the relationship between protective enzymes activity and resistant cultivars are negative correlated, while a positive correlation between the content of MDA and resistance of the cultivars.4. To study the genes expressed differentially in resistance of FM in maize, we constructed four cDNA libraries by suppression subtractive hybridization (SSH) method using the RNAs from the bracts of resistant maize cultivar Bt-1 as well as susceptible maize cultivar Ye478. The obtained cDNA fragments were cloned into pMD 18-T easy vector and checked the recombination efficiency of 4 SSH libraries by PCR. The lengths of PCR products were 0.2-1.0 kb and clones containing no or more than one insert were removed from further investigation. A total of 6,560 (efficiency rate 93%) clones with 3,560 clones from the resistant two libraries in Bt-1 cultivar and 3,000 clones from the susceptible two libraries in Ye478 cultivar were screened and re-examined by PCR. The re-amplified PCR products were dot-blotted on membrane and hybridized with four kinds of DIG-labeled probes. As a result,145 positive clones were obtained and sequenced from all 6560 clones through reverse dot-blot hybridization screening. The DNA sequences of the 145 positive clones were analyzed against GeneBank database, resulting in identification of 93 unique genes.28 cDNA clones were obtained from forward libraries and 12 were obtained from reverse libraries in the resistant cultivar Bt-1.27 subtracted genes were obtained from forward libraries and 26 were obtained from reverse libraries in the susceptible cultivar Ye478.5. Base on the 93 unique genes, the amino acid sequences of 68 genes were identified to exhibit high homology to the genes with function known, while 24 were genes with function unknown. Moreover, one gene did not match any known sequences. These 68 genes were categorized according to the functions of the proteins. The cellular functions of these identified in Bt-1 can be classified into disease defense (8 distinct proteins encoded by 8 ESTs:20.0%), gene destination and transcription (6 distinct proteins encoded by 7 ESTs:17.5%), signal transduction (5 distinct proteins encoded by 5 ESTs:12.5.0%), protein destination and storage (3 distinct proteins encoded by 3 ESTs:7.5%), metabolism (2 distinct proteins encoded by 2 ESTs:5.0%), energy (1 distinct proteins encoded by 1 EST:2.5%), unknown (35.0%including 14 ESTs). The cellular functions of these genes identified in Ye478 can be classified into disease and defense (10 distinct proteins encoded by 13 ESTs:24.5%), gene destination and transcription (8 distinct proteins encoded by 9 ESTs:16.9%), signal transduction (4 distinct proteins encoded by 5 ESTs:9.4%), protein destination and storage (6 distinct proteins encoded by 7 ESTs:13.2%), metabolism (3 distinct proteins encoded by 3 ESTs:5.7%), energy (3 distinct proteins encoded by 3 ESTs: 5.7%), intracellular trafficking (1 distinct proteins encoded by 2 ESTs:3.8%), unknown (22.6%including 12 ESTs).6. Using the GeneChip Maize Genome Array platform, we investigated gene expression changes in the maize bract between resistant inbred Bt-1 and susceptible inbred Ye478 at 96 h post-inoculation with FM. In total, there were 482 unique genes up-regulated more than 1.5 fold in resistant inbred Bt-1 (ANOVA, p<0.05) when compared to mock-inoculated bract tissues. However, only seven of these genes were up-regulated in susceptible inbred Ye478, indicating that the gene expression in the resistant genotype responded strongly to FM. For the genes that were significantly different in both inbreds, we observed that all the seven genes identified in susceptible inbred Ye478 were found in the 482 FM-induced genes of resistant inbred Bt-1. Further analysis of the 482 FM-induced genes indicated that they represented 372 UniGenes and 110 unknown functions based on the bioinformatic analysis. With the information from the genechip, we were able to identify and assign putative function of the FM-induced genes into eight functional categories. The largest category was "defense anti-microbial proteins", including proteins related to elevated disease resistance, which was the most abundant and accounted for 15%of the genes. The second most abundant category was "metabolism and energy" including lipid, carbohydrate and primary metabolism and energy, accounting for 14%of the genes. Other categories included "signal transduction" (12%), "transcriptional regulators" (11%), "reactive oxygen scavengers" (11%), "protein destination" (7%), "transporters (5%), and "stress proteins" (3%). A total of 22%of the genes were "unclassified or with unknown function proteins".7. Molecular annotation system was used to analyze these large number genes differentially expressed from SSH and Genechip FM infection. The results showed that these large reponsive genes were belonged to cellular component, molecular function, biological process with complicated network. In addition,340 single-copy differentially expressed sequences were found from SSH including 93 sequences and Genechip including 482 FM-induced genes after bioinformatics alignment. Then, the the co-localization analysis was performed between the 340 single-copy and ear rot QTL locus which from other researches in the map of maize chromosomes. The result showed that 36 FM-induced genes were located in the Meta-QTLs region and most other genes were closed with the Meta-QTLs. It showed that our results was comformed with others QTLs research conclusion.8. Expression profiles of representative maize ear rot defense-related genes during the time course of infection were indeed confirmed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that expression of genes involved in the FM infection were up-regulated in the resistant maize cultivar as well as susceptible maize cultivar.9. In this research, we investigated the processes involved in maize ear rot upon infection by FM, the time-course infection was observed through SEM to demonstrate pathogen progression in maize bract and the biochemical and physiological enzymes activities were analyzed in the two maize cultivars, Bt-1 and Ye478, completely resistant and significantly susceptible to FM respectively. The FM-responsive genes from both cultivars SSH libraries and genechip data presented here are a valuable resource for further functional genomics studies addressing resistant mechanisms in maize ear rot. Further functional analysis of these responsive genes to FM may provide new insights into the molecular mechanisms of the host defense response. In future studies, the involvement of each gene in FM inducement should be investigated and will help us clarify the process of defense mechanisms acquisition combating invasion in maize ear rot. Such information can be used by breeders for selection of candidate genes, and their transfer to agronomically important maize cultivars.
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