Source: PURDUE UNIVERSITY submitted to NRP
GENETIC, GENOMIC AND PHYSIOLOGICAL UNDERPINNINGS OF MAIZE IMMUNITY TO FUNGAL PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1010573
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Botany & Plant Pathology
Non Technical Summary
Our investigations with the maize - C. carbonum pathosystem have led to a number of milestones in the area of plant-pathogen interactions. However, there are two major aspects of this system that remain unresolved. The first concerns HC-toxin (HCT) that bestows on C. carbonum race 1 a kind of pathogenic potential that stands unprecedented among maize pathogens. Without HCT, C. carbonum is a relatively benign pathogen that survives in nature by largely taking advantage of senescing grass tissues. However, with HCT as a part of its pathogenic arsenal, it turns into a killer (provided maize lacks its shield of HCTR) of exceptional virulence that can decimate every part of the maize plant at every stage of development. The potential threat that HCT poses to its host is further underscored by the fact that the natural mechanism of resistance that evolved in maize to deal with this pathogen is targeted against the toxin itself (Johal and Briggs, 1992). What does HCT do to maize to deserve such an attention? The research proposed herein is expected to generate research tools in the form of mutants and genes to answer this question convincingly.Information and resources generated during this project will also help address the second major unknown about this pathosystem: what underlies the APR behavior of some alleles of hm1? Our observations strongly point in the direction of NADPH as a mediator of APR. If true, the APR alleles of Hm1 could be construed as sensors of NADPH levels in maize plants. The research proposed herein will address this hypothesis using a combination of genetic, genomics, and biochemical approaches and, in addition, expand the network of genetic and metabolic factors capable of impacting maize immunity. The successful outcome of the project will have implications for the design of strategies needed to boost plant immunity.Although the proposed experiments will focus on one well-studied pathosystem, it is likely that the results will also be relevant to other disease interactions (Johal et al, 1995). C. carbonum is a good representative of some of the most serious pathogens of cereal crops. These include C. heterostrophus, which destroyed 15% of the U.S. corn crop in 1970, and C. Victoria, which caused major epidemics of oats in the 1940's in the United States. C. miyabeanus and B. sacchari are major pathogens of rice and sugarcane, respectively. Another parallel between C. carbonum race 1 and these pathogens is that many of them synthesize low molecular secondary metabolites as part of their virulence strategy (Walton, 2005).
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011510104010%
2011510108020%
2011510116020%
2061510104020%
2061510108020%
2061510116010%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1160 - Pathology; 1080 - Genetics;
Goals / Objectives
While the identity and interaction of two key players of the maize-CCR1 pathosystem has been well established, there are two questions that remain unresolved. The first is: How does HCT assist maize colonization by CCR1? Without HCT, CCR1 is a benign pathogen capable of colonizing only the senescing tissues of maize and other grasses (Scheffer et al, 1967). However with HCT as its virulence weapon, CCR1 turns into a maize killer of unprecedented pathogenic potential. Understanding the nature of host mechanisms that HCT subverts to induce susceptibility to CCR1 is one objective of this project.The second objective concerns some alternate forms of the hm1 disease resistance gene that confer immunity in a developmentally-specified fashion. They confer little or no protection at the seedling stage but get better and better with age to become fully efficacious at maturity (Nelson and Ullstrup, 1964). The behavior of these adult plant resistance alleles of hm1 contrasts with that of the wild-type (WT) Hm1 allele that confers complete immunity to CCR1 in every part of the maize plant at every stage of development. Seeking an answer to what contributes to the APR phenotype of certain alleles of hm1 is the second objective of this project.?
Project Methods
Objective 1. How does HCT allow CCR1 to cause disease?One clue comes from the finding that HCT is a potent inhibitor of histone deacetylases (HDACs) (Brosch et al, 1995). Since HDACs are known to be involved in chromatin remodeling, it was thought that HCT would prevent the induction of defense response genes, therefore leaving the host in a susceptible state. However, results from our recent studies are at odds with this hypothesis. By using HCT-minus isolates and exogenously applied HCT on susceptible (hm1hm1) leaves, we found that HCT is able to induce host susceptibility to these nonpathogenic isolates of CCR1 even if the resistance response is already expressed and the pathogen fully contained. Taken together, these studies suggest that HCT induces susceptibility in maize to CCR1 by a mechanism(s) other than simply the suppression of defense response genes.We propose to investigate HCT-induced susceptibility in maize by using a combination of genetic and genomics approaches. The genetic approach will rely on a suppressor/enhancer screen following chemical mutagenesis with EMS. CCR1-susceptible plants will be mutagenized to develop thousands of individual M2 populations, which will be screened for mutants that are no longer susceptible to CCR1. Such CCR1-resistant mutants may have genetic lesions in genes that are either direct targets of HCT or are involved in HCT-mediated induction of susceptibility. All mutants will be selected and characterized in depth with various genetic and molecular biological approaches to clone their underlying genes.We were unable to effectively use this enhancer/suppressor strategy previously because of two problems. First, it is impossible to grow hm1 null plants in the field because of the lethality of the disease caused by CCR1, which kills completely susceptible maize plants no matter where we plant them in the field. Second, the efficiency of the pollen mutagenesis protocol that the corn geneticists are constrained to use is not very high, thereby limiting our ability to carry out mutant hunt screens effectively in this plant species (Candela and Hake, 2008). We have eliminated both of these problems now. A partial loss-of-function mutant of hm1 has been identified in the uniform background of B73 that although is susceptible at the seedling stage can be grown to maturity because of its APR phenotype. And for the enhancer/suppressor screen, we have devised a new strategy that combines multiple rounds of pollen and seed mutagenesis protocols over a couple of generations to enhance mutation rate by 5-10 fold (Dilkes and Johal, 2015). This enhancement in mutagenesis efficiency decreases dramatically the number of M2 plants needed to be screened for mutations, thereby making it practical to conduct the genetic approach.The genomics approaches will rely on using the enhanced analytical power of various profiling technologies such as RNA-seq, proteomics and metabolomics. For proteomics, we will pay special attention to proteins that undergo differential acetylation during disease development. There are two reasons for taking this approach. First, it is becoming increasingly evident that HDACs not only deacetylate histone proteins but also many others, both regulatory and structural (Novell and McMahon, 2010). Interestingly the activities of many of the enzymes of the intermediary metabolism are regulated by their acetylation/deacetylation status, thereby regulating not only the flux through various primary metabolic pathways but also the production of bioenergetic molecules such as ATP and NADPH. Second, the acetylation/deacetylation profile of these enzymes is readily altered by HDAC inhibitors, often compromising the metabolic flux through these pathways (Novell and McMahon, 2010). The proteomics experiments will be conducted in collaboration with Justin Walley of Iowa State University, who has expertise in this area and is conducting mass spectrometry-based proteomics to study post-translational modificationsduring plant-pathogen interactions.For metabolomics analysis, we will also use a pathogen-free surrogate system to look into the identity of HCT-impacted metabolites that are strictly of host origin. This system will comprise elongating roots of germinating maize seeds from both the resistant and susceptible isogenic lines and HCT. In fact, it was on the basis of root growth inhibition assays that HCT was initially identified as a host specific toxin (Comstock and Scheffer, 1973). While HCT was able to suppress the growth of CCR1-susceptible roots at fairly low concentrations, resistant roots requited 100 times more toxin to exhibit equivalent reduction in growth. The design of the metabolomics experiment will include control roots of both susceptible and resistant plants, as well as those which are challenged by N-acetylchitooligosaccharide, a potent elicitor of pattern triggered immunity (PTI) in maize (Schmelz et al, 2014).Objective 2. What underlies the APR behavior of certain alleles of hm1?The phenomenon of adult plant resistance (APR) - wherein the ability of a plant to resist a disease improves with age - operates in several plant pathosystems. Despite the isolation of several APR genes, belonging to both the R and non-R gene class, what underlies this phenomenon remains enigmatic. The maize-CCR1 offers a unique opportunity to look into the mechanistic basis of APR, as there are multiple alleles of hm1 that exhibit an APR phenotype in this pathosystem.One such allele is Hm1A, which we have cloned and characterized. Although the HCTR it encodes differs from the wild-type HCTR (encoded by Hm1) by five amino acids, the Lysine to Histidine change at position 110 seems to be responsible for the APR behavior of Hm1A. This change only weakens the activity of HCTR, as the enzyme can still reduce HCT, albeit at a much reduced rate. Similarly the HCTR encoded by Hm2, an APR duplicate of hm1, also reduces HCT at a much lower level. Hm2 encodes a truncated HCTR that lacks the last 52 amino acids compared to the wild-type HCTR (Chintamanani et al. 2008). Given that one common feature of these two APR forms of hm1 is their partial loss-of-function, this tempted us to consider that the APR behavior of these genes resulted from their weak mutant nature. In support of this idea, we were able to generate three new APR alleles of Hm1 by targeted mutagenesis with EMS. All of these new APR alleles contain missense mutations involving single amino acid substitutions, while a number of other fully-susceptible alleles that were also generated in this mutagenesis experiment all contain drastic mutations such as premature stop codons or splice junction variants.Thus, it is an attribute of the weak mutant alleles of Hm1 to confer APR - but the question is why? We have already ruled out differential transcription or translation to be the reason for their APR behavior, suggesting that something else happens either post-translationally to HCTR or to its activity that renders these alleles APR. That it may have a connection with host metabolism or energetics was suggested by experiments in which the light regime was changed during infection. While the extended light regimes (18h light vs. 6h dark) strengthened APR, the extended darkness (6h light vs. 18h dark) had an opposite effect, delaying both the timing and magnitude of APR. We have also seen APR to weaken under field conditions by stresses that compromise photosynthesis. Based on these observations our working hypothesis is that it is probably the availability of NADPH - a cofactor for HCTR - that orchestrates APR. We will use a combination of genetic and genomics approaches similar to the ones detailed above for objective 1 to address this hypothesis. In addition, we will monitor NADPH at different times of the day and at different stages of growth to address if the levels of NADPH correspond with the APR behavior of hm1-encoded resistance in maize.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:The scientific community at large reached through publications. University students through class teaching in the field of advanced plant pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Much of what was accomplished during this reporting period was the result of MS research by Mark Wanhainen, who graduated in May 2021. After completing MS, Mark took the position of Manager at a large vegetable farm in Idaho. A Ph.D. student - Sendi Jimenez - did a rotation of 6 weeks in the lab exploring why maize silks interact differently with CCR1. It is likely she will return to my lab to pursue her Ph.D. research. Three undergraduate students spent their summer 2021 working on this project. Emily Kuhn did her BS in Agronomy and is now enrolled in an MS program in the Horticulture department but conducting the genetics part of her project on hybrid vigor in corn in my lab. Brook M. Nelson is a BS student in Horticulture. The third student was Cooper M. Nevitt, an undergrad in the Biochemistry department, and he is returning in Spring 2022 to do a research credit course in my lab. was one, and the other was. All three of them worked closely with me helping and learning in field nurseries. The project allowed them to learn many techniques relating to plant pathology and genetics. In addition, they learned how to phenotype plants in the field using various parameters, and how to work in a team-oriented environment. How have the results been disseminated to communities of interest?- Through peer-reviewed publications - Through class teaching - Through interactions with scientists at various meetings - By training the next generation of scientists What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Two research areas addressed were the physiological basis of APR and the impact of autoimmunity on CCR1 interaction with maize. We previously showed that it is the feature of weak alleles of Hm1 to confer APR, and it appears to result from the lack of physiological resilience of maize seedlings to provide sufficient levels of NADPH required by weak Hm1 alleles-encoded mutant HCTRs. Given that the metabolic resilience of foliar tissues is also compromised during the ear fill period, we asked if resistance conferred by APR alleles will show a corresponding decline during this developmental period. Indeed, it does. We found that while maize plants carrying weak mutant alleles of Hm1 turn fully resistant to CCR1 by flowering, they gradually lose their ability to resist during the ear-fill period, with the severity of the disease correlating inversely with the HCTR activity of the APR allele. These results demonstrate that it is not the age of host that dictates APR - as is mostly believed - but the metabolic resilience of the maize plant. CCR1 being a fungal necrotroph, we also tested if it would be able to colonize autoimmune maize mutants undergoing HR cell death spontaneously. Necrotrophic pathogens are believed to use any kind of cell death to their advantage, including HR. This was reported with Botrytis cinerea in Arabidopsis and C. victoriae in oats. This is not the case however with CCR1, even though it is closely related to C. victoriae. CCR1 was unable to colonize maize cells dying from HR in Rp1-D21, an autoimmune mutant, suggesting that not all necrotrophic pathogens are equipped to manipulate HR cell death for host colonization. CCR1 was also not discouraged from colonizing Rp1-D21 autoimmune mutants lacking the hm1 disease resistance gene. These plants were as susceptible to CCR1 as those without Rp1-D21, even though they had their defense responses already induced. These observations imply that induction of susceptibility of maize to CCR1 is not mediated by the suppression of host defenses by HC-toxin, as has been presumed.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2021 Citation: Thesis Title: Title: New insights from the Northern Leaf Spot disease of maize challenge the prevailing views of adult plant resistance and the necrotrophic mode of fungal parasitism. Author: Mark P. Wanhainen Program: MS
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Karre S, Kim SB, Kim BS, Khangura RS, Sermons SM, Dilkes B, Johal G, Balint-Kurti P. 2021. Maize Plants Chimeric for an Autoactive Resistance Gene Display a Cell-Autonomous Hypersensitive Response but Non-Cell Autonomous Defense Signaling. Mol Plant Microbe Interact. 34: 606-616. doi: 10.1094/MPMI-04-20-0091-R.


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The scientific community at large reached through publications. University students through classroom teaching in the field of advanced plant pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project was instrumental in the career development of Amanpreet Kaur, a graduate student who successfully finished all the requirements for her Ph.D. degree in July 2020. She is now a postdoctoral fellow in our biochemistry department, working on a project that aims to integrate genetics and computational biology to explore maize growth and development. Mark Wanhainen is a master's student and his research project is focusing on adult plant resistance to CCR1. He plans to finish his degree in Spring 2021. Two undergrad students that worked on this project during summer 2020 were Ms. Madylin Xiao Schaider and Brenna Porsch, both undergraduate students in our department. The project allowed them to learn many techniques relating to plant pathology and genetics. In addition, they learned how to phenotype plants in the field using various parameters, and how to work in a team-oriented environment. A postdoctoral fellow, Dr. Bong-Suk Kim, was also an active participant on this project. He left the lab in May 2020 and was hired by the department to help coordinate plant growth facilities. How have the results been disseminated to communities of interest?- Through peer-reviewed publications - Through classroom teaching - Through interactions with scientists - By training the next generation of scientists What do you plan to do during the next reporting period to accomplish the goals?Now that we have demonstrated that DILI is a real phenomenon, we will conduct RNA-seq analyses to explore the genetic networks and mechanisms that are at play during DILI. Another project concerns the dynamics of adult plant resistance (APR) that is being pursued by Mark Wanhainen, a graduate student in the lab. Observations have been made that indicate that the protection provided by weak APR alleles of Hm1 breaks down during the ear fill period. This again brings host metabolism and physiology in the realm of immunity in maize.

Impacts
What was accomplished under these goals? In the 2019 report, we highlighted the discovery of an intimate link between host metabolism and immunity in maize. We demonstrated that conditions and treatments that inhibit photosynthesis cause maize seedlings to succumb to disease by diverse fungal pathogens. We dubbed this phenomenon dark-induced loss of immunity (DILI), because it was first witnessed in maize seedlings incubated under extended darkness following inoculation with an avirulent isolate of CCR1. Interestingly, the exogenous administration of sucrose alone was able to rescue maize seedlings from undergoing DILI. Intrigued by this unique link between photoassiminemia and maize immunity, we decided to screen an EMS-mutagenized B73 M3 library for mutants that resisted infection by CCR1 during 48-hour dark incubation. One mutant was identified that turned out to be allelic to the sucrose export defective1 mutant, designated sxd1. Although the primary defect in sxd1 lies in vitamin E biosynthesis, one consequence of this defect is the callose-mediated plugging of plasmodesmata in bundle sheath cells. This prevents the export of sucrose and other photosynthates, thereby leading to their buildup in the leaf tissue. Two other maize mutants with a similar phenotype in sugar partitioning are tdy1 (tie-dyed1) and su1 (sucrose transporter1). And as with sxd1, they also resisted DILI during our assay conditions, clearly confirming the causal link between host metabolism and immunity in maize. These results are very significant, especially in the light of some recent observations we have made about maize plants deficient in jasmonic acid (JA) or salicylic acid (SA). While these two hormones have been found to orchestrate most aspects of immunity in Arabidopsis and many other plants, their impact on the interaction of maize with CCR1 is negligible. Taken together, these findings suggest that the immune system in maize may be wired differently compared to what we have learned over the past 2 to 3 decades from the model plant Arabidopsis. These findings also indicate that one way to boost maize immunity would be to enhance the photosynthetic capacity and vigor of maize seedlings.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Khangura RS, Venkata BP, Marla SR, Mickelbart MV, Dhungana S, Braun DM, Dilkes BP, Johal GS. 2020. Interaction Between Induced and Natural Variation at oil yellow1 Delays Reproductive Maturity in Maize. G3 (Bethesda) 10:797-810. doi: 10.1534/g3.119.400838.PMID: 31822516.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Khangura RS, Johal GS, Dilkes BP. 2020. Variation in Maize Chlorophyll Biosynthesis Alters Plant Architecture. Plant Physiol. 2020 Sep;184(1):300-315. doi: 10.1104/pp.20.00306. Epub 2020 Jul 8.PMID: 32641472
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jagtap AB, Vikal Y, Johal GS. 2020. Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize. Int J Mol Sci. 2020 Oct 6;21(19):7386. doi: 10.3390/ijms21197386.PMID: 33036291.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The scientific community at large reached through publications and poster presentations. University students through class teaching in the field of advanced plant pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A new graduate student, Mark Wanhainen, joined our group as master's student in January 2019. Mark's research focus is to investigate whether APR in maize to CCR1 is an irreversible or reversible phenomenon. This he is addressing under both field and greenhouse conditions by asking what happens to APR during the ear-fill stage of plant development. An exchange PhD. Student - Ashok Jagtap - from Punjab Agricultural University, India also joined the lab in January 2019 to stay until December 29, 2019. One of the projects that Ashok is involved in is exploring the effect of temperature - both hot and cold - on APR. Two undergrads students worked on this project during the summer months of 2019. Jacob Lee, an undergrad in the Biological Sciences department at Purdue, was one, and the other was Charandeep (Chuck) Singh, an Ivy Tech Community College student. Both Jacob and Chuck helped with the project in the summer genetic nurseries and worked closely with the PI and a postdoctoral fellow Dr. Bong-Suk Kim. The project allowed them to learn many techniques relating to plant pathology and genetics. In addition, they learned how to phenotype plants in the field using various parameters, and how to work in a team-oriented environment. How have the results been disseminated to communities of interest?- Through peer-reviewed publications - Through class teaching - Through interactions with scientists at various meetings - By training the next generation of scientists What do you plan to do during the next reporting period to accomplish the goals?Having discovered DILI/SILI and established many its physiological and pathological features of this phenomenon, we would like to seek mutants that fail to undergo SILI. For this project, we will screen an M3 population of B73 that was generated by a new EMS mutagenesis protocol, termed next generation mutagenesis (NextGEM). This mutagenesis approach combined seed and pollen mutagenesis in a single generation to generate a large library of the M2 seed that contains mutations in a heterozygous state at a very high density, at a frequency of ~1x10-2. Six hundred and eighty plants from this M2 library were self pollinated to generate M3 ears that have been shelled and packaged individually. Sixteen plants from each M3 family will be inoculated with CCR1 and incubated under extended darkness (48 h) to identify DILI tolerant mutants.

Impacts
What was accomplished under these goals? Two important discoveries were reported in the 2018 report. First, we demonstrated that the APR nature of the maize Hm1 alleles is the consequence of their partial loss of function. Hm1 alleles that are fully function confer complete protection in every part of the plant and at every stage of development. Maize plants containing fully null hm1 alleles are susceptible throughout plant development. In contrast, plants that have weak alleles of Hm1 are susceptible at the seedling stage but turn resistant at maturity. The second discovery was that the inability of APR alleles to confer resistance to CCR1 was due to the lack of metabolic resilience of the maize plants at the seedling stage. This limited capacity of metabolic buffering results in NADPH deficiency during the night hours. Given that the function of Hm1 encoded HCTR depends on having NADPH as a cofactor, reduction of NADPH below a threshold level compromises the HC-toxin-reducing activity of the enzymes encoded by the APR alleles. This hypothesis was validated by growing maize seedlings under different light regimes. Seedlings that were grown under 20 hours of continuous light had their APR alleles confer effective seedling resistance, while the seedlings grown under 20 hours of continuous darkness had a negative impact on APR. During one of these light-regime manipulation experiments, our growth chamber broke down and the plants experienced a continued darkness of about 60 h. To our amazement, even our control plants containing the WT Hm1 gene - which are normally immune to CCR1- developed susceptible lesions under these conditions of extended darkness. This result was reproducible and showed that under prolonged darkness maize seedlings lose their ability to mount an immune response to CCR1. A number of follow-up experiments were done that established the following features of this dark-induced loss of immunity (DILI). It takes as little as 36 hours of continuous darkness to induce DILI. Not only does DILI prevent the induction of immunity, it also causes immunity to break down if it is already induced and the pathogen is contained. DILI is not unique to CCR1, as resistance to other pathogens, including C. heterostrophus, Excerohilum turcicum and Colletotrichum graminicola, is also abolished by extended darkness. DILI even allowed the rice blast pathogen (Magnaporthe grisea) to infect maize, highlighting that DILI erodes immunity at the most basic level. DILI is a strictly a feature of maize seedlings, as plants become more and more resistant to undergo DILI beyond 4-weeks of age. The entire plant has to be under darkness for DILI to manifest, and DILI is not induced if only a part of the plant is subjected to darkness. DILI does happen under dim light, and occasional flashes of bright light fail to revert DILI. These observations ruled out the involvement of light signaling as a mechanism responsible for DILI induction. That it may have a metabolic basis was suggested by the suppression of DILI by exogenously supplied sugar. In line with these results, the chemical inhibition of photosynthesis by the herbicide DCMU also led to DILI. These results suggest that it is the loss of photosynthates that result in DILI. One mechanism by which it could happen is by the induction of the starvation response that can interfere both with the induction and maintenance of immunity. If true, the acronym for DILI will have to be changed to SILI, for starvation-induced loss of immunity.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: He Y, Karre S, Johal GS, Christensen SA, Balint-Kurti P. 2019. A maize polygalacturonase functions as a suppressor of programmed cell death in plants. BMC Plant Biol. 2019 Jul 15;19(1):310. doi: 10.1186/s12870-019-1897-5.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Khangura RS, Marla S, Venkata BP, Heller NJ, Johal GS, Dilkes BP. 2019. A Very Oil Yellow1 Modifier of the Oil Yellow1-N1989 Allele Uncovers a Cryptic Phenotypic Impact of Cis-regulatory Variation in Maize. G3 (Bethesda). 2019 Feb 7;9(2):375-390. doi: 10.1534/g3.118.200798.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The scientific community at large reached through publications. University students through class teaching in the field of advanced plant pathology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research work of two graduate students over the past several years led to the findings highlighted here. Both students, Kevin Chu and Sandeep Marla received their doctoral degrees in the last two years, and both are doing their postdoctoral research currently. Two undergrads students who worked on this project in the past year are Samantha McLoughlin and Nicole Woloshuk, both undergraduate students at Purdue. In addition, our SROP student from Puerto Rico Myrrh N Perez Ruiz was also involved in the project. SROP (Summer Research Opportunities Program) is a gateway program to graduate school that helps prepare underrepresented minorityundergraduates for graduate study through intensive research experiences with faculty mentors and enrichment activities. All three of the undergrads helped with the project in the summer genetic nurseries and worked closely with the PI and a postdoctoral fellow Dr. Bong-Suk Kim. The project allowed them to learn many techniques relating to plant pathology and genetics. In addition, they learned how to phenotype plants in the field using various parameters, and how to work in a team oriented environment. How have the results been disseminated to communities of interest?- Through peer-reviewed publications - Through classteaching - Through interactionswith scientists at various meetings What do you plan to do during the next reporting period to accomplish the goals?The first goal will beseek how the level of NADPH changes over time daily in maize seedlings up to 3 weeks of age, and at weekly intervals throughout the life of a maize plant. The second goal will be to explore various chemicals or metabolites that may boost the metabolic status of the host to allow APR alleles of hm1 to confer seedling resistance.

Impacts
What was accomplished under these goals? Last year we reported on the characterization of an allele of hm1 - Hm1A - that provides effective resistance against CCR1 only at maturity. The detailed characterization of this APR allele predicted Hm1A to be partial loss-of-function allele of the Hm1 gene. This prediction was confirmed by generating two new alleles of Hm1 by targeted mutagenesis with EMS, a chemical mutagen. Like Hm1A, these APR alleles (Hm1-2 and Hm1-3) also had a partial loss of their function. However, the question of why do the weak alleles of Hm1 conferred remained unresolved. In this report we provide significant insights into this question. First, we showed that different APR alleles differed in the strength of their resistance phenotypes, with Hm1-2 being the strongest and Hm1-3 being the weakest among our collection of 3 APR alleles. Second, we developed a biochemical assay that demonstrated that the HCTR (HC-toxin reductase) enzymes that these APR alleles encode are relatively weaker than the HCTR encoded by the wild-type Hm1 gene. Additionally, the relative strength of the HCTR activity encoded by various APR alleles closely matched the strength of their resistance phenotypes. The strength of the HCTR encoded by Hm1-2 was the highest, followed by those encoded by Hm1A and Hm1-3, respectively. Their transcriptional and translational activities however stayed constant throughout plant development, suggesting that the weak activity of their HCTRs was somehow responsible for their APR phenotype. We went on to show that this was true. Given that the activity of HCTR depends on the availability of cofactor NADPH, our results indicated that maize seedlings have relatively limited amount of NADPH and that it falls below the threshold required for the weak HCTRs encoded by the APR alleles of hm1. Consistent with this hypothesis, the resistance phenotype of the APR alleles improved under conditions that enhanced the photosynthetic output of the seedlings and weakened under conditions that inhibited photosynthesis. These results imply that the metabolic demand for the weak resistance alleles is relatively high and is easily upset by conditions that impair the metabolic wellbeing of the host. Among other things, this work suggests that there is a rather close connection between immunity to diseases and host metabolism.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Marla SR, Chu K, Chintamanani S, Multani DS, Klempien A, DeLeon A, Bong-Suk K, Dunkle LD, Dilkes BP, Johal GS. 2018. Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1. PLoS Pathog. 14:e1007356. doi: 10.1371/journal.ppat.1007356.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sindhu A, Janick-Buckner D, Buckner B, Gray J, Zehr U, Dilkes BP, Johal GS. 2018. Propagation of cell death in dropdead1, a sorghum ortholog of the maize lls1 mutant. PLoS One. 13:e0201359. doi: 10.1371/journal.pone.0201359.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:- The scientific community at large reached through presentations, posters and abstracts at scientific meetings, and through invited seminars at universities. - Students through teaching a class on Advanced Plant Pathology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student involved in the project was Kevin Chu, who defended his thesis defense in on November 29, 2016 but stayed on in the lab as a temporary postdoc until the end of May 2017. The title of his thesis was Dissecting the multifaceted relationship between maize and Cochliobolus carbonum race 1. Kevin is currently a postdoc at the Danforth Plant Science Center in St. Louis. Rhiana Ragheb, a junior high school student, helped with the project in the summer and worked closely with the PI. The project allowed Ms. Ragheb to learn many techniques relating to plant pathology and genetics. In addition, she learned how to phenotype plants in the field using various parameters, and how to work in a team oriented environment. How have the results been disseminated to communities of interest?- Through thesis publication: Thesis author: Kevin Chu Title:Dissecting the Multifaceted Relationship between Maize and Cochliobolus carbonum race 1 Thesis advisor: Gurmukh S. Johal - Through seminar presentations Gurmuh. S. Johal. Ohio State University, Columbus. April 10, 2017. Topic. Characterization of a lesion mimic mutant in maize uncovers a homolog of RIN4 and its guard NLR. - Through meeting abstracts and posters One meeting abstract and poster was presented at the 59th Maize Genetics Meeting held in St. Louis, Missouri, from March 9 to 12, 2017. Abstract Chu, Kevin; Best, Norman, DeLeon, Alyssa; Rhodes, David; Dilkes, Brian; Johal, Guri. HC-toxin causes massive transcriptional and metabolic changes in maize during Cochliobolus carbonum race 1 infection. Poster 121, Page 125. What do you plan to do during the next reporting period to accomplish the goals?Clone and characterize various APR genes in te maize-CCR1 pathosystem to figure out the mechanism(s) underlying adult plant resistance. Will also generate maize isogenic lines in the B73 background containing various APR alleles varying in strength and onset. Publish a paper on our recent findings showing that one requirement for APR is to have partial loss-of-function or weak mutations in the Hm1 gene.

Impacts
What was accomplished under these goals? We were able to make significant inroads into understanding the mechanistic basis of APR in this pathosystem. We cloned and characterized Hm1A, one of the naturally occurring APR alleles at the hm1 locus. The predicted HCTR that it encodes differs from that the wild-type HCTR (encoded by Hm1) by five amino acids. However, only the Lysine to Histidine change at position 110 (L110H) seems to be solely responsible for the APR behavior of Hm1A. The other four amino acid changes were ruled out because one of the lines (a landrace from Bolivia) shared all four of these changes with Hm1A but was still resistant to CCR1. Moreover, the Lysine residue mutated in HM1A is absolutely conserved not only in all the orthologs of Hm1 found in all grasses tested, but also in the maize dihydroflavonol reductase (DFR) enzyme, which is also an NADPH-dependent reductase with significant homology to HCTR. The L110H change in HM1A only has partial effect on the activity of HCTR, and the enzyme can still reduce HCT, albeit at a much reduced rate. Similarly the wild-type allele at the hm2 locus, which is a homeologous duplicate of hm1, confers APR to CCR1. Like Hm1A, Hm2 also appears to be a partial loss-of-function mutant as it lacks the last 52 amino acids of the wild-type HCTR. Given that one common feature of these two APR alleles is their partial loss-of-function, this led us to reason that the APR behavior of these genes might result from their weak mutant nature. In support of this idea, we were able to generate three new APR alleles from the WT Hm1 by a targeted mutagenesis screen involving ethyl methanesulfonate (EMS), a chemical mutagen. All of these new APR alleles contain missense mutations leading to conservative amino acid substitutions, while a number of other fully-susceptible alleles that were also generated in this mutagenesis experiment contain drastic mutations such as premature stop codons or splice junction variants. These results convincingly demonstrate that the APR phenotype of maize genes to CCR1 (i.e., to confer resistance at maturity but not at the seedling stage) is an attribute of the weak mutant nature of these APR genes. However, the question of why do they behave in this fashion remains unresolved.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Kevin Chu. Dissecting the Multifaceted Relationship between Maize and Cochliobolus carbonum race 1. Thesis advisor: Gurmukh S. Johal