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ABSTRACT Understanding how pathogens infect different tissue types and how plants mount tissue‐specific defences against bacterial pathogens is critical for improving disease resistance.Xanthomonas vasicolapv.vasculorum(Xvv) is a bacterial pathogen of maize that exhibits both vascular and nonvascular lifestyles, offering a unique opportunity to dissect host–pathogen interactions across tissue types. Here, we combined fluorescence microscopy, dual RNA‐seq and functional genetics to characterize the interactions between Xvv and maize, both vascular and nonvascular. Using distinct inoculation methods, we reveal temporally and spatially divergent colonization patterns and transcriptional responses in resistant (B73) and susceptible (CML333) maize lines. We show, using transposon insertion lines, that a malectin‐like receptor kinase (ZmCrRLK1L19) confers resistance to vascular disease. The gene colocalizes with a major Xvv vascular resistance QTL. Xvv modulates motility and virulence gene expression depending on tissue context; flagellar motility is essential for mesophyll colonization but is dispensable in the xylem. These findings reveal mechanisms underlying tissue‐specific resistance and pathogen tissue tropism, providing a foundation for improving broad‐spectrum disease resistance in cereal crops. 

Exserohilum turcicum, the causal agent of northern corn leaf blight (NCLB) and sorghum leaf blight (SLB), exhibits host-specific interactions at both the species and genotype levels. While maize- and sorghum-adapted strains are generally restricted to their respective hosts, further specificity is observed in maize through gene-for-gene interactions with Ht resistance genes. In this study, we characterized the infection process of E. turcicum across three interaction types: unadapted (sorghum specific strain on maize, or maize specific strain on sorghum), adapted-susceptible (each strain on its respective host), and adapted-resistant (maize specific strain on the Ht1 maize differential). We collected quantitative data for the early colonization phenotypes using epifluorescence and confocal fluorescence microscopy. There were significant differences between adapted and unadapted interactions at multiple infection steps. In the unadapted interactions, the pathogen formed more appressoria, and the host had more dead cells under the appressoria, suggesting a stronger hypersensitive response (HR). Adapted-susceptible interactions exhibited higher levels of xylem and mesophyll penetration and colonization than unadapted and adapted-resistant interactions. Moreover, successful xylem penetration without colonization was observed in unadapted interactions, while adapted resistant interactions demonstrated both xylem penetration and colonization without mesophyll colonization. Progeny strains adapted to one or both hosts confirmed these patterns and showed no fitness cost associated with dual host adaptation. These findings provide new insights into the basis of host specificity and resistance to E. turcicum and highlight the potential for discovering novel resistance mechanisms by studying unadapted interactions across host species. 

How host genotype shapes pathogen tissue tropism remains poorly understood. Vascular and nonvascular tissues represent distinct habitats within a plant for bacteria to colonize. Host plants often utilize different mechanisms to defend themselves against vascular and nonvascular pathogens, and mechanisms of resistance employed by the host can vary by organ. Xanthomonas vasicola pv. vasculorum ( Xvv) is an emerging bacterial maize pathogen, and this pathosystem offers an opportunity to study how host resistance differs in response to the vascular and nonvascular lifestyles exhibited by a single bacterial phytopathogen. We used different inoculation techniques to induce vascular and nonvascular disease and evaluated maize populations using both techniques to map resistance to vascular and nonvascular disease caused by Xvv. Xvv can colonize both vascular and nonvascular tissues, depending on the genotype. Different inoculation techniques can be used to induce vascular or nonvascular colonization. Independent loci control variation in resistance to Xvv during vascular and nonvascular pathogenesis. We confirmed the role of those regions in resistance to vascular and nonvascular infection. This study offers insights into how host resistance shapes how bacterial pathogens adapt to both vascular and nonvascular lifestyles. We show that host genotype can dictate which tissues a pathogen can infect. This system can serve as a model to understand tissue-specific host resistance to plant pathogens and tissue specificity in pathogens. 

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 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. 

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 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. 

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. 

Abstract Gibberella ear rot (GER) of maize, caused byFusarium graminearum, poses a serious threat to human and animal safety as mycotoxins are deposited in the grain during fungal colonization and are hazardous to human and animal health. Currently, no completely resistant germplasm has been identified, and the underlying mechanisms of resistance remain unclear. In this study, we evaluated three near‐isogenic line (NIL) populations for resistance to GER—NC344 × H100 (DRIL), B73 × Oh43 (nNIL), and B73 × teosinte (tNIL)—across multiple environments. The recurrent parents, H100 and B73, were moderately resistant to GER. NC344 is susceptible to GER. Oh43 is moderately resistant to GER. The teosinte donor parent, PI 384071, had unknown resistance to GER. We employed two inoculation methods—kernel injection and silk channel injection—to assess their impact on genotype response to F. graminearum. The inoculation method did not significantly affect genotype response, although kernel inoculations produced more consistent disease levels. We identified lines with significantly increased susceptibility to GER compared to their recurrent parent in each population. We employed quantitative trait locus (QTL) mapping to identify markers associated with GER in the NC344 × H100 population. We identified QTL on chromosomes 4, 5, and 9. We highlight a large region on chromosome 5 that may harbor important alleles for GER resistance and for resistance to other ear rots. This study underscores the utility of NILs in dissecting the genetic basis of GER resistance and provides valuable resources for future fine mapping, gene discovery, and resistance breeding. 

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.