Location: Corn Host Plant Resistance Research2012 Annual Report
1a. Objectives (from AD-416):
Identify genes and proteins that enhance resistance to Aspergillus (A.) flavus infection and aflatoxin accumulation in maize: (1) isolate and determine functions of genes associated with resistance to A. flavus infection and aflatoxin accumulation in maize, (2) compare expression patterns of genes associated with resistance in maize cultivars with varying levels of resistance or susceptibility to A. flavus/aflatoxin, (3) determine abiotic and biotic factors that affect the expression of genes associated with resistance to A. flavus/aflatoxin.
1b. Approach (from AD-416):
Maize cultivars with varying levels of resistance or susceptibility to A. flavus infection and aflatoxin accumulation will be grown in field plots. Developing ears will be inoculated with toxin-producing and non-toxin-producing strains of A. flavus at predetermined times after pollination, and tissue will be collected from both inoculated and non-inoculated ears at various intervals throughout the growing season. Plants will be monitored for response to drought and heat stress. Maize and fungal proteins, RNA, and DNA will be extracted from tissue samples. Expression profiles of different tissues collected at various times from resistant and susceptible maize genotypes grown under different conditions will be compared using electrophoresis, microarrays, micro-RNA, and other appropriate proteomic or genomic techniques. Genes and proteins identified in these studies will be developed as molecular markers for enhancing resistance to aflatoxin accumulation in maize hybrids.
3. Progress Report:
The goals and objectives continued to be the identification of genes associated with resistance to Aspergillus (A.) flavus infection and aflatoxin accumulation in maize in general. Special goals for this period were set to focus on a few parental inbred lines (Mp715, Va35, Mp313E) and some of their derivative recombinant inbred lines (RILs) (Mp718 and Mp719) that were selected against the resistance trait. A very efficient protocol was established to solve the major computational obstacles in the identification of significantly differentially expressed genes that have critical effects from high throughput gene expression data. This protocol was effective in both microarray experiments and quantitative real-time polymerase chain reaction (qRT-PCR) experiments and will now be used routinely. Accomplishments achieved through this improved computational method include: 1) Sixteen candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. For example, a gene encoding glycine-rich ribose nucleic acid (RNA) binding protein 2 (AI664980) was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene (TC231674) was found to be involved in the host resistance in Mp313E. 2)The following are some details on the findings: TC231674 is the highest expressed found in Mp313E samples. TC231674 encodes a NUP85-like gene that is a part of a sub-complex of the nuclear pore complexes (NPCs) embedded in the nuclear envelope. The function of NPCs is for the transport of RNA and other macromolecules from nucleus to cytoplasm. TC237311 and BM379345 are second highest in expression. TC237311 encodes the heat shock protein HSP101. This protein acts as a molecular chaperone to disaggregate mis-folded proteins. BM379345 encodes a metallothionein like protein (MTLP) that is involved in the binding and detoxification of heavy metal ions. BE050050 and TC238832 comprise the next level in expression. BE050050 has no annotation available. TC238832 encodes a lecithin cholesterol acyltransferase (LCAT)-like gene. Other highly expressed genes include BM498943, BM078796, and TC207503. BM498943 encodes ethylene responsive protein (ETHRP) that belongs to the universal stress protein family. BM078796 encodes small heat shock protein HSP26. TC207503 encodes a prenylated rab acceptor (PRA1) family protein. Prenylated Rab PRA1 proteins are small transmembrane proteins that regulate vesicle trafficking. The highest expressed genes found in Va35 were AI664980 and BG266083. AI664980 belongs to the glycine-rich RNA binding protein family (GRBP2) that has RNA binding domains (RBD) commonly found in proteins involved in post-transcriptional gene expression processes. BG266083 encodes alpha-crystallin-type of stress-induced small heat shock proteins (HSP18a). TC234808 and TC223372 were also highly expressed in Va35. TC234808 is in the ribosomal protein L30 family (RPL30). The ribosomal protein L30 has pre-mRNA splicing regulatory activity to its own transcript and plays a key role in the assembling of the ribosomal subunits. TC223372 is a cinnamoyl-CoA reductase (CNCR2) that catalyses the lignin pathway. Other highly expressed genes found in Va35 included TC218605, TC226528, and AI065864. TC218605 encodes phytochrome A (PHYA). TC226528 encodes a ureide permease (UPS) that transport and recycle organic nitrogen for nucleotide synthesis. AI065864 belongs to the exonuclease-endonuclease-phosphatase (EEP) domain superfamily. 3) In the experiment involved in Mp715, Va35, and the derivative RILs, eleven genes (out of 40) were found to be differentially expressed in Mp718 and Mp719 compared to Va35 by qRT-PCR. More candidate genes (40 more) are being tested in lab. One gene, TC231674, was found significantly highly expressed in both Mp718 and Mp313E. The obstacle of this experiment was that Mp715 plants didn’t grow well in the field in 2011, therefore we are missing one parent control for the data analysis. New Mp715 plants are being planted in field in 2012. 4) These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation. Genes significantly associated with resistance are being further evaluated by USDA-ARS scientsts at Mississippi State. Polymorphisms in TC238832 and TC231674 sequences were found to diffentiate resistant and susceptible genotypes in a preliminary test.