THE GENETIC BASIS OF MAIZE RESPONSE TO ARBUSCULAR MYCORRHIZAL FUNGI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1029193
• Grant No.: 2022-67013-38264
• Cumulative Award Amt.: $1,247,490.00
• Proposal No.: 2022-07492
• Project Start Date: Sep 15, 2022
• Project End Date: Sep 14, 2026
• Grant Year: 2022
• Program Code: [A1171]- Plant Biotic Interactions

Project Director
Sawers, R.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505

Performing Department
(N/A)

Non Technical Summary
OVERVIEWSince first colonizing land, plants have relied on beneficial associations with microorganisms to gain access to soil nutrients. Today's major crop species retain this capacity to form symbioses with soil microbes, although these interactions may not be optimized for agricultural systems. The agroecosystem exposes these ancient associations to a high-input environment that differs greatly from that in which they evolved. Furthermore, modern crop varieties are the product of intensive artificial selection under these same high-input conditions. To fully realize the potential of microbial interactions in agriculture, we require a better understanding of when, why and how they will have the greatest benefit. In this project, we will characterize the drivers of variation in maize (Zea mays; corn) response to arbuscular mycorrhizal (AM) fungi. Specifically, we will 1) identify variety-specific host molecular and physiological responses to inoculation with diverse AM fungi and 2) map the genetic architecture of AM host response in the context of the abiotic and biotic environment of the cultivated field. We will compare maize lines previously identified to differ in AM host response using a combination of fine-scale analysis in controlled conditions and genetic mapping in the field. For field experiments, we will use a design we have developed to selectively incorporate plant AM-incompatibility genes into our mapping populations to estimate AM effects.INTELLECTUAL MERITThe balance of cost and benefit in symbioses is dynamic, and there is a fine line between mutualism and parasitism. The prevailing hypothesis states that agricultural intensification has reduced the benefit crop plants can derive from microbial interactions. Although fundamental to the application of plant-microbial associations, this hypothesis has been difficult to address. Manipulation of microbial communities in the field at the scale required for meaningful evaluation of yield components is logistically challenging. This project will use a novel mapping strategy to estimate the impact of a major crop-microbial mutualism under cultivation. In conjunction with molecular and physiological characterization, including profiling of the AM fungal and broader microbial communities, our experiments will distinguish dependence on symbiosis from benefit - concepts that have previously been difficult to pin down. We will assess trade-offs between the performance of symbiotic and non-symbiotic plants with implication as to the extent to which breeding for high-input agriculture can also be aligned with maintaining the capacity of plants to benefit fully from AM associations.BROADER IMPACTSAs society demands more sustainable agriculture, it is important to develop robust methodologies to evaluate the viability and potential benefits of alternatives to conventional practice. This work will contribute substantially to our understanding of the role of crop-microbial mutualisms and the degree to which the breeding process needs to take account of these interactions if we are to benefit fully from them. The inherent appeal of the integration of beneficial microbes into farming systems presents an excellent opportunity to engage the broader community in learning about biotic interactions, plant genetics, and the future of sustainable agriculture. We will use AM fungi incompatible maize varieties to provide a striking visual demonstration of the importance of symbiotic soil microbes to crop performance. We will collaborate with existing PSU extension and outreach partners to communicate findings from this project to the agronomic research, farming, and broader community. We will participate in an existing range of activities, presenting annually in PSU's principal agricultural outreach show and a local agricultural fair. We will develop and host an annual tour of our research field and facilities for Middle School students. We will engage with the Penn State Arboretum to develop a living exhibit of AM compatible and incompatible varieties growing side-by-side and available for the public. We will build on project team experience and existing collaborations to produce on-line and printed content explaining the importance of beneficial plant-microbe interactions to young audiences

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1510	1080	50%
201	1510	1010	30%
102	4099	1010	20%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn; 4099 - Microorganisms, general/other;

Field Of Science
1010 - Nutrition and metabolism; 1080 - Genetics;

Keywords

cereal root symbiosis; mycorrhizal benefit

maize

mycorrhizae

Goals / Objectives
PROJECT AIM: Characterize the drivers of variation in maize response to AM fungiWe will use i) a bottom-up approach (GOAL1) to characterize physiological, morphological, anatomical,and molecular differences in Oh43 and B73 responses to AM fungi and, ii) a top-down approach(GOAL2) to map regions of the genome associated with variation in AM symbiotic outcome. Together,these approaches will provide a holistic view of differences in AM response. The intersection of thesetwo approaches will identify candidate genes for further functional analysis and application (Fig. 1).GOAL1: Identify host genotype-specific molecular and physiological responses to controlledinoculation with diverse AM fungi. We will grow Oh43 and B73 lines in controlled conditions, withand without inoculation with a range of AM fungal species. We will evaluate growth and nutrient levels,root-internal and external fungal structures, root architecture and anatomy, AM partner choice, andpatterns of whole-genome transcription to identify host genotype specific effects.GOAL2: Map the genetic architecture of AM host response in the context of the abiotic and bioticenvironment of a cultivated field. We will map the genetic architecture of AM host response in thefield using a population generated by crossing each of B73 and Oh43 to an AM fungi-incompatiblesentinel line. We will evaluate phenology, morphology, yield components, leaf and grain nutrient levels,and composition of the root and rhizosphere microbiome. We will determine the impact of AM fungi onagronomically important plant traits and assess trade-offs between growth with or without AMsymbiosis. We will characterize the field microbiome and variation in the AM fungal community.

Project Methods
GOAL1. Characterize the molecular and physiological signature of AM host responseThe maize line Oh43 is highly responsive to the broadly studied AM fungi R. intraradices and F. mosseae. In GOAL1, we will characterize AM host response in Oh43 through comparative transcriptomics; assess the consistency of the Oh43 response with respect to a range of AM fungal species; investigate host choice of partners when several fungal species are present; and correlate host response with variation in root anatomy. We will compare Oh43 with the reference line B73. For all experiments in Goal 1, we will grow plants in pots in a greenhouse system using a sand-clay substrate. Plants will be fertilized with Hoagland solution adjusted to reducedphosphate, following previously established experimental conditions. We will harvest batches of plants at 4 and 8 weeks for sample collection in accordance with previous morphological and molecular studies of the progression of the symbiosis. Plant growth response will be calculated as the difference in above ground biomass between inoculated and non-inoculated plants. Fungal colonization will be determined by microscopy and quantitative PCR using fungal- and plant- specific primers. In addition, root segments will be retained for analysis using Laser Ablation Tomography, as described in EXP1.3. Root-external fungal hyphae will be quantified by microscopic inspection of bulk substrateand measurement of the phospholipid fatty acid biomarker PLFA 16.1ω5 using gas chromatography.GOAL2. Map the genetic architecture of AM host response in the fieldWe will map host AM response over two years at the PSU research farm under standard management without additional AM inoculation. Preliminary observation of inbred AM-I sentinel stocks has indicated that there is a significant effect of native AM fungi on maize growth and performance in this location. To characterize host response in the field, we have developed a mapping strategy in which half of our families are homozygous for a mutation conferring incompatibility to AM fungi, while the other half are homozygous wild-type and compatible with the fungus. This design allows estimation of 1) the overall impact of AM fungi on a given trait by taking the average difference between compatible and incompatible families, and 2) the interaction between allelic differences underlying trait variation(QTL) and the compatibility phenotype, describing the genetic architecture of AM host response. Importantly, this analysis distinguishes variation in AM dependence and benefit. QTL mapping will identifyAM compatibility x QTL interaction consistent with variation in dependence or benefit. Rank-changing effects willindicatetrade-offs: the best plant genotype in the absence of AM fungi can bedifferent fromthe best genotype in the presence of AM fungi, with implications for breeding for optimized use of AM fungi in agricultural systems.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:Results have been communicated through teaching, seminars, publication in peer reviewed scientific journals and group activities to the student and academic community. Changes/Problems:The availabilty of a collecton of B73xOh43 near isogenic lines (BC5; B73 recurrent)has allowed their inclusion in EXP1.2. This modification also nicely complements the allele specific expression experiment EXP1.1: briefly, cis-acting variants identified in EXP1.1 can be followed in the corresponding NILs that carry Oh43 alleles at such loci in the B73 background. What opportunities for training and professional development has the project provided?PSU project members Melanie Perryman (Lab Manager), Patrick Sydow (PhD student PSU Agricultural and Environmental Plant Science program; Burghardt-Sawers Group), Courtney Tharp (PhD student Plant Biology program; Dini-Andreote Group) and Sergio Perez-Limon (PhD student PSU Agricultural and Environmental Plant Science program; Sawers Group) have gained experience in maize genetics, genotypic analysis, characterization of AMF and evaluation of field grown plants. Five Penn State undergraduates have also participated in the project durng the reporting period, both in the laboratory and the field. How have the results been disseminated to communities of interest?Results have been presented in a publication (Yu et al. 2024), and presented in poster form at theInternational Molecular Mycorrhiza Meeting, Cambridge, UK, September 2023 and the International Conference on Mycorrhizae, Manchester, UK, August 2024. What do you plan to do during the next reporting period to accomplish the goals?GOAL1. Characterize the molecular and physiological signature of AM host response EXP1.1 Variation in host transcriptional response to AM fungi We will complete the analysis of available RNAseq data, identifying candidate genes and assessing the capacity of the transcriptome to predict AM outcome in our field data. EXP1.2. Host response stability and choice of AM fungal partners We will evaluate Oh43, B73, W22 in the greenhouse following inoculation with different AMF species.We will complete analysis of B73xOh43 NILs, including cross-analysis with the Oh43 v B73 expression data from EXP1.1 EXP1.3. Evaluating the interplay between root anatomy and mycorrhizal colonization We will sequence available root/rhizosphere DNA samples from the MAGIC population; we will analyse the existing root anatomical images. GOAL2. Map the genetic architecture of AM host response in the field EXP2.1 Field evaluation of host response in an AM-C_AM-I mapping population We now have genotypic data and complete phenotypic data for a number of traits, assessed over two years. We will conduct mapping analyses, using a number of approaches to incorporate AM status, as well as soil analysis covariants. In parallel, we will add to the phenotypic dataset by completing evaluation of material harvested Fall 2024.

Impacts
What was accomplished under these goals? GOAL1. Characterize the molecular and physiological signature of AM host response EXP1.1 Variation in host transcriptional response to AM fungi We have complete extraction of 100 samples for the B73 v Oh43 allele-specific expression transcriptome analysis. Sequencing libraries have been prepared by the Penn State genomics core facility and sequenced. Initial quality control has indicated the data to be of high quality. We have begun the analysis to identify differential gene expression and cis-acting expression variation. EXP1.2. Host response stability and choice of AM fungal partners We have selected two representative fungi (Rhizophagus irregularis and Funneliformis mosseae) and used these to inoculate a collection of ~40 B73-Oh43 near isogenic lines. This slight modification will allow us to look at consistency of response across two fungi but also complements well EXP1.1. EXP1.3. Evaluating the interplay between root anatomy and mycorrhizal colonization We continue analysis of anatomy and microbiome data from the maize MAGIC population. GOAL2. Map the genetic architecture of AM host response in the field EXP2.1 Field evaluation of host response in an AM-C_AM-I mapping population We have completed characterization ears samples from the 1350 rows of the 2023 field evaluation, including taking morphological measurements, total ear weight, total grain weight and 50 grain weight. In Summer 2024, we planted a second replicate of the field experiment, again growing 3 complete blocks of 450 families. The in-season traits plant height and flowering time have been collected for 2024, such that we now have two full years of data to begin genetic mapping. Ears and stover will be harvested for further evaluation in the Fall to complete the field trait dataset in the Spring 2025.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Yu, B., Zhou, C., Wang, Z., Bucher, M., Schaaf, G., Sawers, R., Chen, X., Hochholdinger, F., Zou, C. and Yu, P. (2024). Maize zinc uptake is influenced by arbuscular mycorrhizal symbiosis under various soil phosphorus availabilities. The New phytologist. DOI: 10.1111/nph.19952

Progress 09/15/22 to 09/14/23

Outputs
Target Audience:Results have been communicated through teaching, seminars, publication in peer reviewed scientific journals and group activities to the student and academic community Changes/Problems:In line with reviewer comments, we have reduced emphasis on EXP2.2Characterization of the root microbiome in AM-C_AM-I F2:3 mapping populations. This question is partially incorporated into EXP1.3Evaluating the interplay between root anatomy and mycorrhizal colonization, where we will now take advantageof an existing maize MAGIC population experiment/sampling by adding AMF data to address this question. We have advanced well with EXP2.1Field evaluation of host response in an AM-C_AM-I mapping population, being in a position to conduct the first field evaluation in year 1 of the project. We have focused on the Oh43 x W22 cross for this aspect of the work. What opportunities for training and professional development has the project provided?Project members Melanie Perryman (Lab Manager Sawers Group), Li Meng (Postdoc Sawers Group), Jagdeep Sidhu (Postdoc Sawers Group), Patrick Sydow (PhD student PSU Agricultural and Environmental Plant Science program; Burghardt-Sawers Group), Courtney Tharp (PhD student Plant Biology program; Dini-Andreote Group) and Sergio Perez-Limon (PhD student PSU Agricultural and Environmental Plant Science program; Sawers Group) have gained experience in maize genetics, genotypic analysis, characterization of AMF and evaluation of field grown plants. Several undergraduates have also participated in the project both in the laboratory and the field. How have the results been disseminated to communities of interest?Results have been communicated through teaching, seminars, publication in peer reviewed scientific journals and group activities. The project has produced 1 peer reviewed articles and 3 external seminars during the reporting period. Li, M., Perez-Limon, Sergio, Ramirez-Flores, M Rosario, Barrales-Gamez, Benjamin, Meraz-Mercado, M. A., Ziegler, G. R., Baxter, I., Olalde-Portugal, Victor, & Sawers, R. (Author) (2023). Mycorrhizal status and host genotype interact to shape plant nutrition in field grown maize (Zea mays ssp. mays).Mycorrhiza, 1--14. Sawers, R. (Invited Speaker; September, 2023). "Mycorrhizal Response in Maize," ENSA project meeting, Cambridge University, Cambridge, UK. Sawers, R. (Poster Presentation; September, 2023). "Mycorrhizal Response in Maize," International Molecular Mycorrhiza Meeting, Cambridge University, Cambridge, UK. Sawers, R. (Keynote Speaker; October, 2023). "The contribution of the root system to local adaptation of Mexican native maize," XX National Plant Biochemistry and Molecular Biology Congress, 3rd Meeting of the Mexico Section of the American Society of Plant Biologists, 13th Mexico-USA Plant Biology Symposium, Oaxaca, Mexico. What do you plan to do during the next reporting period to accomplish the goals?GOAL1. Characterize the molecular and physiological signature of AM host response EXP1.1 Variation in host transcriptional response to AM fungi We will obtain RNA sequencing data and form allele-specific analysis. EXP1.2. Host response stability and choice of AM fungal partners We will evaluate Oh43, B73, W22 in the greenhouse following inoculation with different AMF species. EXP1.3. Evaluating the interplay between root anatomy and mycorrhizal colonization We will use DNA sequencing to characterize the AMF and broader microbial community of maize MAGIC root and rhizosphere samples. GOAL2. Map the genetic architecture of AM host response in the field EXP2.1 Field evaluation of host response in an AM-C_AM-I mapping population We will continue post-harvest evaluation of the 2023 field experiment. Tissue has been harvested from all families and submitted for genotyping (RAPID genomics) using a custom set of ~3000 selected SNP markers. Genotypic data should be available early 2024 for genetic mapping and QTL analysis. We will conduct further field evaluation Summer 2024, with the design informed by the initial 2023 results.

Impacts
What was accomplished under these goals? GOAL1. Characterize the molecular and physiological signature of AM host response EXP1.1 Variation in host transcriptional response to AM fungi We have established soil and growth conditions for Oh43, W22 and B73 inbred lines and Oh43xW22 and Oh43x B73 F1 hybrids, with and without inoculation with AMF, in the greenhouse. We have collected all samples from the allele specific transcription experiment, harvesting roots at 3 and 7 week time points. Samples for RNA sequencing were selected from broader sampling based on representative biomass and levels of AMF colonization. RNA has been extracted and checked for quality for timepoint 1. We are in the process of completing extraction of RNA for timepoint 2 before submitting all 100 samples for library preparation and sequencing at the Penn State genomics core facility. EXP1.2. Host response stability and choice of AM fungal partners We are growing up inoculum for 5 species of AMF for an experiment early 2024. EXP1.3. Evaluating the interplay between root anatomy and mycorrhizal colonization We have collected root samples from a diverse maize MAGIC population for which root anatomy data is also available. We are in the process of extracting root/rhizosphere DNA for characterization of microbial/specific AMF community by molecular methods. GOAL2. Map the genetic architecture of AM host response in the field EXP2.1 Field evaluation of host response in an AM-C_AM-I mapping population We have generated and bulked 450 F2:3 families for "sentinel" mapping from the cross W22 x Oh43. We Single Nucleotide Polymorphism (SNP) genotyping to identify wild-type families and those fixed for AMF incompatible mutations. In Summer 2023, we field-evaluated 280 wild-type families, 60 castor families, 40 pollux families and 40 ccamk families (2:1 AMF-compatible to AMF-incompatible) over 3 replicates using a design incorporating AMF-C or AMF-I subplots. Single row sub-sub-plots of 14 plants were scored for flowering time, height and stover dry weight. Ears have been collected for measurement of yield components and element analysis. Individual plants were excavated from selected (~10% total) of sub-sub-plots and roots samples collected to score AMF colonization. High resolution soil maps were generated by sampling and analysis at over 90 positions across the field. Initial analyses have revealed significant relationships between soil nutrient availability and plant phenotypic response to AMF compatibility. Tissue has been harvested from all families and submitted for genotyping (RAPID genomics) using a custom set of ~3000 selected SNP markers. Genotypic data should be available early 2024 for genetic mapping and QTL analysis.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Li, M., Perez-Limon, Sergio, Ramirez-Flores, M Rosario, Barrales-Gamez, Benjamin, Meraz-Mercado, M. A., Ziegler, G. R., Baxter, I., Olalde-Portugal, Victor, & Sawers, R. (Author) (2023). Mycorrhizal status and host genotype interact to shape plant nutrition in field grown maize (Zea mays ssp. mays). Mycorrhiza, 1--14.