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ABSTRACT It is more than 40 years since the era of transgenic plants began and more than 30 years after the cloning of the first plant disease resistance genes. Despite extensive progress in our mechanistic understanding and despite considerable sustained efforts in the commercial, nonprofit, academic and governmental sectors, the prospect of commercially viable plant varieties carrying disease resistance traits endowed by biotechnological approaches remains elusive. The cost of complying with the regulations governing the release of transgenic plants is often cited as the main reason for this lack of success. While this is undeniably a substantial hurdle, other transgenic traitshavebeen successfully commercialised. We argue that a significant portion of the challenges of producing crop varieties engineered for disease resistance is intrinsic to the trait itself. In this review, we briefly discuss the main approaches used to engineer plant disease resistance. We further discuss possible reasons why they have not been successful in a commercial context and, finally, we try to derive some lessons to apply to future efforts. 

Abstract Exserohilum turcicum causes northern corn leaf blight and sorghum leaf blight. While the same species cause disease in both crops, the strains are host-specific. Here, we report the sequence and de novo annotated assemblies of one sorghum- and one maize-specific E. turcicum strain. The strains were sequenced using the PacBio Sequel II system. The total genome length for both assemblies was between 44 and 45 Mb with N50 of ∼2.5 Mb. Ninety-eight percent of the Benchmarking Universal Single-Copy Orthologs (BUSCO) for both assemblies had complete status. The estimated number of genes was 11,762 and 12,029 in the sorghum- and maize-specific isolates, respectively. Funannotate, EffectorP, SignalP, and transcriptome data were used to create functional annotation of each genome. The whole-genome comparison identified ten large-scale inversions and three translocations between the maize- and sorghum-specific strains, along with homologous genes and gene duplications. RNA was sequenced from the maize- and sorghum-specific isolate 10 days post-inoculation in maize and sorghum and from axenic cultures. Gene expression data from planta and axenic growth experiments were compared for each strain. Candidate host-specificity genes were identified by combining results from whole-genome comparison, synteny analysis, gene annotations, and transcriptome data. Overall, this study identified several candidate host-specificity genes that provide insights into E. turcicum interaction with its hosts. 

SUMMARY In this study, we characterized a panel of 1264 maize near‐isogenic lines (NILs), developed from crosses between 18 diverse inbred lines and the recurrent parent B73, referred to as nested NILs (nNILs). In this study, 888 of the nNILs were genotyped using genotyping‐by‐sequencing (GBS). Subsequently, 24 of these nNILs, and all the parental lines, were re‐genotyped using a high‐density single nucleotide polymorphism (SNP) chip. A novel pipeline for calling introgressions, which does not rely on knowing the donor parent of each nNIL, was developed based on a hidden Markov model (HMM) algorithm. By comparing the introgressions detected using GBS data with those identified using chip data, we optimized the HMM parameters for analyzing the entire nNIL population. A total of 2969 introgressions were identified across the 888 nNILs. Individual introgression blocks ranged from 21 bp to 204 Mbp, with an average size of 17 Mbp. By comparing SNP genotypes within introgressed segments to the known genotypes of the donor lines, we determined that in about one third of the lines, the identity of the donors did not match expectation based on their pedigrees. We characterized the entire nNIL population for three foliar diseases. Using these data, we mapped a number of quantitative trait loci (QTL) for disease resistance in the nNIL population and observed extensive variation in effects among the alleles from different donor parents at most QTL identified. This population will be of significant utility for dissecting complex agronomic traits and allelic series in maize. 

Abstract The pattern‐triggered immunity (PTI) response is triggered at the plant cell surface by the recognition of microbe‐derived molecules known as microbe‐ or pathogen‐associated molecular patterns or molecules derived from compromised host cells called damage‐associated molecular patterns. Membrane‐localized receptor proteins, known as pattern recognition receptors, are responsible for this recognition. Although much of the machinery of PTI is conserved, natural variation for the PTI response exists within and across species with respect to the components responsible for pattern recognition, activation of the response, and the strength of the response induced. This review describes what is known about this variation. We discuss how variation in the PTI response can be measured and how this knowledge might be utilized in the control of plant disease and in developing plant varieties with enhanced disease resistance. 

Abstract Foliar diseases of maize are among the most important diseases of maize worldwide. This study focused on 4 major foliar diseases of maize: Goss's wilt, gray leaf spot, northern corn leaf blight, and southern corn leaf blight. QTL mapping for resistance to Goss’s wilt was conducted in 4 disease resistance introgression line populations with Oh7B as the common recurrent parent and Ki3, NC262, NC304, and NC344 as recurrent donor parents. Mapping results for Goss’s wilt resistance were combined with previous studies for gray leaf spot, northern corn leaf blight, and southern corn leaf blight resistance in the same 4 populations. We conducted (1) individual linkage mapping analysis to identify QTL specific to each disease and population; (2) Mahalanobis distance analysis to identify putative multiple disease resistance regions for each population; and 3) joint linkage mapping to identify QTL across the 4 populations for each disease. We identified 3 lines that were resistant to all 4 diseases. We mapped 13 Goss’s wilt QTLs in the individual populations and an additional 6 using joint linkage mapping. All Goss’s wilt QTL had small effects, confirming that resistance to Goss’s wilt is highly quantitative. We report several potentially important chromosomal bins associated with multiple disease resistance including 1.02, 1.03, 3.04, 4.06, 4.08, and 9.03. Together, these findings indicate that disease QTL distribution is not random and that there are locations in the genome that confer resistance to multiple diseases. Furthermore, resistance to bacterial and fungal diseases is not entirely distinct, and we identified lines resistant to both fungi and bacteria, as well as loci that confer resistance to both bacterial and fungal diseases. 

Maize is a globally important staple that is used as food for human and animal consumption, fuel, and other industrial applications. Pathogens affect all stages of the plant life cycle and every plant organ, and lead to significant yield losses. An integrated strategy incorporating cultural and chemical management practices, as well as development of resistant plant varieties, is needed to prevent yield losses due to plant diseases. Large numbers of breeding material must be screened to develop pathogen-resistant maize varieties. Inoculation methods must be high-throughput to accommodate the large screening experiments. Additionally, there needs to be an extensive understanding of the plant–pathogen interaction to use a targeted biotechnology-based approach, which takes advantage of knowledge of the system to engineer resistance. To evaluate germplasm for breeding and biotechnology approaches, inoculation methods must replicate natural infection, and disease severity must be rated consistently to accurately screen germplasm or gather data on pathogens of interest. Here, we review inoculation and rating methods for Gibberella ear rot, seedling blight caused byGlobisporangium ultimumvar.ultimum, and Goss's wilt that are efficient and high-throughput. We also introduce fluorescence microscopy techniques for leaf samples infected withExserohilum turcicum, the causal agent of northern corn leaf blight. These pathogens all cause significant yield losses, and in particular, Gibberella ear rot is associated with the accumulation of harmful mycotoxins. Understanding how pathogens cause disease and how plants defend against attack is a major goal of maize pathology studies and critical for developing integrated management strategies. 

Maize is an important food and fuel crop globally. Ear rots, caused by fungal pathogens, are some of the most detrimental maize diseases, due to reduced grain yield and the production of harmful mycotoxins. Mycotoxins are naturally occurring toxins produced by certain fungal species that can cause acute and chronic health issues in humans and animals that consume mycotoxin-contaminated grain. Pathogens can infect the developing ear through silks, or through wounds in the ears produced by pests. Plants naturally develop genetic resistance to pathogens. The maize genes involved in resistance to the pathogen may be different, depending on whether the ear was infected via silks or wounds. To differentiate between these two forms of resistance, natural infections can be reproduced by injecting inoculum through the silk channel, or by producing wounds using a needle, and introducing inoculum directly onto developing ears. Our protocol describes a technique used to inoculate developing maize ears withFusarium graminearum, one of the fungal species that causes ear rot. We describe both silk channel and side needle inoculation techniques. Our protocol uses a backpack inoculator for both methods of infection, allowing for high-throughput inoculations, which are necessary for large field experiments. After harvest, the ears are visually rated on a percentage of disease scale. The protocol results in quantitative data that can be used for research on elucidating genetic resistance to fungal pathogens to assist breeding selections, and to understand plant–pathogen interactions of ear rots in maize. 

Maize is a globally important grain crop that is important for food and fuel. Northern corn leaf blight, caused byExserohilum turcicum, is an important fungal foliar disease of maize that is highly prevalent and causes yield losses globally. Microscopy can be used to visualize plant–fungal interactions on a cellular level, which enables pathology and genetics studies. Host resistance and isolate aggressiveness can be characterized at different stages of disease development, which enables a more detailed understanding of the pathogenesis process and host–pathogen interactions. Our protocol outlines an efficient, cost-effective method for stainingE. turcicumtissue on inoculated maize leaves and visualizing samples using a compound fluorescence microscope. This protocol uses KOH treatment followed by aniline blue staining, which stains glucans present in plant and fungal cell walls, and samples are visualized using fluorescence microscopy. Quantitative data about fungal structures including the conidia, hyphal structures, and appressoria, the structures formed to push through the plant leaf surface after conidia have germinated, can be obtained from the images generated using this technique. Visualization of these structures can help pathologists understand plant–pathogen interactions for maize andE. turcicum. This method has advantages over other methods because the stain is less toxic than other available stains, samples can be processed in a more high-throughput manner than other protocols, and the required supplies are relatively inexpensive. 

Maize significantly contributes to food and fuel production. Yields can be reduced due to foliar diseases, which reduce photosynthetic leaf area. The bacterial foliar disease Goss's wilt (caused byClavibacter nebraskensis) can cause significant yield losses in susceptible maize varieties.C. nebraskensiscan infect leaves through wounds and colonize the vascular tissue of the leaf. We present a protocol that replicates this process with the use of a “clapper” with pins on one end to create wounds and a sponge soaked in inoculum on the other end, which allows for efficient field inoculations of maize leaves. Disease severity is then rated on a percentage scale multiple times over the season to generate an area under disease progress curve (AUDPC). Genetic host resistance is one of the most effective forms of foliar disease control in maize, as there are few effective forms of chemical control for bacterial diseases that affect maize. Screening for resistance in diverse germplasm, or for fine mapping a specific resistance gene, requires inoculating large populations in the field for obtaining phenotypic data. Our high-throughput protocol allows for large-scale disease evaluations and is useful for finding forms of genetic resistance or to understand plant–pathogen interactions of bacterial foliar pathogens. 

The southern corn leaf blight epidemic of 1970 caused estimated losses of about 16% for the U.S. corn crop, equivalent to about $8 billion in current terms. The epidemic was caused by the prevalence of Texas male sterile cytoplasm ( cms-T), used to produce most of the hybrid corn seed planted that year, combined with the emergence of a novel race of the fungus Cochliobolus heterostrophus that was exquisitely virulent on cms-T corn. Remarkably, the epidemic lasted just a single year. This episode has often been portrayed in the literature and textbooks over the last 50 years as a catastrophic mistake perpetrated by corn breeders and seed companies of the time who did not understand or account for the dangers of crop genetic uniformity. In this perspective article, we aim to present an alternative interpretation of these events. First, we contend that, rather than being caused by a grievous error on the part of the corn breeding and seed industry, this epidemic was a particularly unfortunate, unusual, and unlucky consequence of a technological advancement intended to improve the efficiency of corn seed production for America's farmers. Second, we tell the story of the resolution of the epidemic as an example of timely, meticulously applied research in the public sector for the public good.