Corn improvement
Developing Biomarkers for Host Plant Susceptibility to Aspergillus Infection.
The goal of this phase of the project is to develop biomarkers that can predict the risk of mycotoxin contamination even before infection actually occurs. We will identify biomarkers indicative of stress before silking that correlate with subsequent mycotoxin contamination. When integrated into management programs, these biomarkers will be able to alert producers to crop stress, providing time to respond with a biocontrol agent or chemical protection. Corn breeders will be able to use the same set of markers in screening programs.
Many phenological studies have focused on the impact of heat and drought stress on kernel numbers and ear growth, including some studies that compared performance of hybrids. These studies have identified a window of ~3 weeks prior to silking as being the most critical for ear growth; stresses applied to plants during this window of corn development can have irreversible effects on ear health. Windham et al found that environmental stress during this period also enhanced susceptibility to A. flavus and contamination with aflatoxin. In this objective, greenhouse experiments will replicate heat and drought stress under controlled conditions. We will measure diverse stress response parameters (e.g., changes in gene expression and metabolite accumulation) during the pre-silking transition to maturity and correlate these parameters with aflatoxin and fumonisin accumulation.
Experiments are being conducted at Purdue University in adjacent greenhouse rooms with in-ground beds. Maize hybrid DKC68-05 are planted at standard field population with full factorial experimental design to test the individual and combined effects of heat (day/night) and water stress. Watered plants in the non-heat-stressed room serve as unstressed controls. Drought and heat stress are applied four weeks prior to silking, with standard watering regimes resumed after pollination. One week after silking, ears are silk-channel inoculated with A. flavus or F. verticillioides. Ear rot severity is then measured at ear maturity, as are aflatoxin and fumonisin levels.
During this period, data and sample collection begin one week after stress is applied, and continue until three weeks after silking. Plants (genrally ~180/treatment) are tagged and followed individually throughout the experiment. At seven-day intervals (six time points), tissue samples and photosynthesis data are collected from the upper-most fully expanded leaves. CO2 fixation, transpiration and leaf temperature are also measured three times during the day. On separate sets of plants, tissue samples are collected at mid-day and stored at -80°C. For metabolite and RNA-seq analyses, three biological replicates are analyzed, consisting of five plants each. We examine two metabolic components in leaf tissues – soluble sugar and free amino acids – with established GC/MS protocols. For RNAseq, total RNA is extracted and sequenced, with a depth of at least 10 million reads per biological rep. Sequence data is then aligned to the maize genome.
Preliminary PCA (principal component analysis) of the transcriptomic data are used to filter out noninformative genes, and to identify genes that show statistically significant correlations with both stress conditions and mycotoxin levels. The overall analysis of this data is being done at Purdue and Texas A&M.Once a gene set is identified whose expression pattern correlates with predisposing environmental stress and mycotoxin contamination in mature kernels, real-time PCR primers will be developed for each of the marker genes. A subsequent greenhouse experiment will be conducted on several hybrids to validate the markers and eliminate those that are unique to a genotype. At the same time, Co-PIs in TX and NC will collect leaf samples from field studies, and send them to Purdue for analysis. By year 4, we anticipate having predictive markers for plant stress that can be integrated into management programs.
Generation and evaluation of transgenic (RNAi) lines of corn for mycotoxin reduction.
Our goal is to generate transgenic corn that inhibits mycotoxin production by host-induced gene silencing (HIGS). Our aim is to produce 50 unique HIGS constructs to silence specific fungal genes via interfering RNA (RNAi), and to express each construct in corn. The sequence of research activities is to
a) Select key genes in A. flavus and F. verticillioides that regulate mycotoxin biosynthesis during kernel colonization
b) Silence candidate fungal genes in vitro to identify the most promising candidates for transgenic silencing
c) Create transgenic corn lines expressing RNAi constructs targeting fungal genes of interest
d) Select candidate transgenic corn lines through testing under laboratory, greenhouse, and field conditions; and
e) Optimize transgenic resistance through gene pyramiding.
Targeting fungal genes involved in kernel colonization or general fungal viability would create a strong selection pressure and thus would not be ideal for durable resistance. Therefore, our guiding criterion is to select genes/pathways that are linked to mycotoxin biosynthesis, yet are dispensable in the kernel environment. Initially, we will target well-studied genes involved in mycotoxin biosynthesis including FUM1, FUM21, aflR, and laeA. We will target hexokinases and genes involved in starch metabolism, which are critical for mycotoxin production. Sequence similarity of these orthologs will allow us to target some genes simultaneously in both species with a single RNAi construct. We also employ network pathway modeling described to identify and select novel regulatory targets.
A. flavus and/or F. verticillioides can be transformed with hairpin RNA vectors corresponding to gene targets. Mycotoxin production is assessed in the resulting transformants in 7-10 days. Candidate fungal genes that show promise in this simple validation assay (i.e., a consistent, substantial reduction in mycotoxin biosynthesis) will be incorporated via Agrobacterium into Hi II immature zygotic corn embryos. Regenerable type II calli will be subcultured and fertile transgenic plants recovered. Regenerating plantlets (primary transgenics; R0) will be used to generate R1 seed to establish transgenic corn lines.
Kernels from each transgenic line will be evaluated for fungal colonization and mycotoxin accumulation in vitro. Transgenic lines that significantly inhibit mycotoxin production compared to the control lines will be selected for further evaluation in greenhouse tests. Transgenic lines that continue to show reductions in mycotoxin will be evaluated thoroughly in field plots.
Because of the complex nature of this disease we do not expect full resistance to mycotoxin contamination to be conferred by any single transgene. However, lines that consistently show even partial reduction of mycotoxin contamination may be extremely valuable, to increase resistance by pyramiding different transgenes to convey an additive or even synergistic reduction in mycotoxin accumulation. Additionally, transgene pyramiding could convey simultaneous resistance to aflatoxin and fumonisin accumulation. To this end, after evaluating resistance conveyed by each single-gene transgenic line, we will initiate a series of crosses in which 2-5 distinct transgenes will be incorporated into a single hybrid. These hybrids will be evaluated for resistance to mycotoxin accumulation as described above.
By the end of the five-year project, we will evaluate at least 50 unique RNAi transgenes for resistance to accumulation of aflatoxins and fumonisins when expressed in corn. We anticipate identifying lines that consistently express high levels of resistance to mycotoxin accumulation. This research will advance our knowledge on HIGS and its application in the control of plant diseases. Furthermore, transgenes with demonstrated efficacy can be tested in other crop species susceptible to aflatoxin and fumonisin contamination.