Gene Ht1 Research Articles

The northern corn leaf blight resistance gene Ht1 encodes an nucleotide-binding, leucine-rich repeat immune receptor.

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E.turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E.turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3.

Alleles of a wall-associated kinase gene account for three of the major northern corn leaf blight resistance loci in maize.

Northern corn leaf blight, caused by the fungal pathogen Setosphaeria turcica (anamorph Exserohilum turcicum), is one of the most devastating foliar diseases of maize (Zea mays). Four genes Ht1, Ht2, Ht3 and Htn1 represent the major sources of genetic resistance against the hemibiotrophic fungus S. turcica. Differential maize lines containing these genes also form the basis to classify S. turcica races. Here, we show that Ht2 and Ht3 are identical and allelic to the previously cloned Htn1 gene. Using a map-based cloning approach and Targeting Induced Local Lesions in Genomes (TILLING), we demonstrate that Ht2/Ht3 is an allele of the wall-associated receptor-like kinase gene ZmWAK-RLK1. The ZmWAK-RLK1 variants encoded by Htn1 and Ht2/Ht3 differ by multiple amino acid polymorphisms that particularly affect the putative extracellular domain. A diversity analysis in maize revealed the presence of dozens of ZmWAK-RLK1 alleles. Ht2, Ht3 and Htn1 have been described over decades as independent resistance loci with different race spectra and resistance responses. Our work demonstrates that these three genes are allelic, which has major implications for northern corn leaf blight resistance breeding and nomenclature of S. turcica pathotypes. We hypothesize that genetic background effects have confounded the classical description of these disease resistance genes in the past.

Diversity of Expression Types of Ht Genes Conferring Resistance in Maize to Exserohilum turcicum.

Northern corn leaf blight (NCLB) is an important leaf disease in maize (Zea mays) worldwide and is spreading into new areas with expanding maize cultivation, like Germany. Exserohilum turcicum, causal agent of NCLB, infects and colonizes leaf tissue and induces elongated necrotic lesions. Disease control is based on fungicide application and resistant cultivars displaying monogenic resistance. Symptom expression and resistance mechanisms differ in plants carrying different resistance genes. Therefore, histological studies and DNA quantification were performed to compare the pathogenesis of E. turcicum races in maize lines exhibiting compatible or incompatible interactions. Maize plants from the differential line B37 with and without resistance genes Ht1, Ht2, Ht3, and Htn1 were inoculated with either incompatible or compatible races (race 0, race 1 and race 23N) of E. turcicum. Leaf segments from healthy and inoculated plants were collected at five different stages of infection and disease development from penetration (0–1 days post inoculation - dpi), until full symptom expression (14–18 dpi). Symptoms of resistance responses conveyed by the different Ht genes considerably differed between Ht1 (necrotic lesions with chlorosis), Ht2 (chlorosis and small lesions), Ht3 (chlorotic spots) and Htn1 (no lesions or wilt-type lesions). In incompatible interactions, fungal DNA was only detected in very low amounts. At 10 dpi, DNA content was elevated in all compatible interactions. Histological studies with Chlorazol Black E staining indicated that E. turcicum formed appressoria and penetrated the leaf surface directly in both types of interaction. In contrast to incompatible interactions, however, the pathogen was able to penetrate into xylem vessels at 6 dpi in compatible interactions and strongly colonized the mesophyll at 12 dpi, which is considered the crucial process differentiating susceptible from resistant interactions. Following the distinct symptom expressions, resistance mechanisms conferred by Ht1, Ht2, Ht3, and Htn1 genes apparently are different. Lower disease levels and a delayed progress of infection in compatible interactions with resistant lines imply that maize R genes to E. turcicum are associated with or confer additional quantitative resistance.

Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants.

Exserohilum turcicum (sexual stage Setosphaeria turcica) is the hemibiotrophic causal agent of northern leaf blight of maize and sorghum. This study aimed to identify the genes involved in host colonization during the biotrophic and necrotrophic phases of infection. It also aimed to identify race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted before inoculation and at 2, 5, 7, and 13 days post-inoculation (dpi). Biological replicates were pooled per time point for each race and sequenced. A bioinformatics pipeline was used to identify candidate effectors, and expression was validated for selected candidates. Fungal biomass was positively correlated with the percentages of E. turcicum reads mapped, which were low at early time points (2–7 dpi) with a significant increase at 13 dpi, indicating a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. AVRHt1 is the putative E. turcicum effector recognized by the maize resistance gene Ht1. Consistent with this, AVRHt1 was expressed in planta by race 23N, but transcripts were absent in race 13N. In addition, specific transposable elements were expressed in 23N only. Genes encoding the virulence-associated peptidases leupeptin-inhibiting protein 1 and fungalysin were expressed in planta. Transcriptional profiles of genes involved in secondary metabolite synthesis or cell wall degradation revealed the importance of these genes during late stages of infection (13 dpi). A total of 346 expressed candidate effectors were identified, including Ecp6 and proteins similar to the secreted in xylem (SIX) effectors common to formae speciales of Fusarium oxysporum, SIX13 and SIX5. Expression profiling of Ecp6 and SIX13-like indicated a peak in expression at 5 and 7 dpi compared to 2 and 13 dpi. Sequencing of SIX13-like from diverse isolates of E. turcicum revealed host-specific polymorphisms that were mostly non-synonymous, resulting in two groups of SIX13-like proteins that corresponded to the maize or sorghum origin of each isolate. This study suggests putative mechanisms whereby E. turcicum causes disease. Identification of the candidate effector SIX13-like is consistent with the infection mode of E. turcicum through the xylem of susceptible hosts.

Occurrence and Distribution of Physiological Races of Exserohilum turcicum in Ontario, Canada.

Northern corn leaf blight (NCLB) caused by Exserohilum turcicum is the most common and economically significant fungal leaf disease of corn in Ontario, Canada. During the past 10 years in Ontario, severity and incidence of NCLB have increased, possibly owing to the appearance of new races. Several races have been identified in various parts of the world; however, information on occurrence and distribution of races in Ontario is lacking. In the current study, 677 single conidial isolates of E. turcicum were isolated from 687 symptomatic leaf samples collected between 2012 and 2016. These isolates were evaluated for pathogenicity on six corn differential inbreds (A619, A619Ht1, A619Ht2, A619Ht3, A632Htn1, and H102Htm1) under controlled environmental conditions and then grouped into 17 physiological races (0, 1, 2, 3, M, N, 12, 1M, 1N, 3M, 13M, 12N, 13N, 1MN, 12MN, 13MN, 123MN) based on the reaction of the inbreds to infection (resistant or susceptible). Four races (0, 1M, 1N, and 1MN) were most frequent, with an isolation frequency of 13, 10, 12, and 41%, respectively. Seventy-six percent of the isolates were virulent on more than one Ht resistance gene, with 2.4% (16 isolates) virulent on all five Ht resistance genes used in this study. Further analysis of the distribution of races in four regions over the years revealed that the occurrence and distribution of the races changed with time in Ontario. Overall, the frequency of virulence of the 677 isolates screened on the differentials with resistance genes Ht1, Ht2, Ht3, Htm1, and Htn1 varied from 6 to 81% (Ht1 81%, Ht2 6%, Ht3 12%, Htm1 64%, and Htn1 64%). Virulent isolates produced fewer lesions on the Htm1 differential, and smaller lesions that were slower and having less sporulation on the Htn1 differential, compared with infection of the differentials with Ht1, Ht2, and Ht3 resistance genes. Virulence frequency also changed within the four geographical regions of Ontario, with fewer isolates virulent on all resistance genes in eastern Ontario compared with southern and western Ontario. Isolates from southern Ontario had greater virulence frequency against Ht1 and Htm1, whereas isolates from western Ontario were more frequently virulent on Ht1 and Htn1. The information generated in this study on the distribution of E. turcicum races in Ontario corn will help growers to select appropriate hybrids with required resistance genes and will assist seed companies in deploying resistance genes in corn hybrids across the province or within a particular region.

The Physical Location of the Gene Ht1 (Helminthosporium Turcium Resistance1) in Maize (Zea Mays L.)

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two RFLP markers, umc22 and umcl22, which are in linkage group 2 in the maize genetic map. The gene ht1 (Helminthosporium turcium resistance 1) is linked closely to umc22 with 4.3 cM on one side and umcl22 with 1.2 cM on the other side. In this study, the FISH results showed the two tested markers were located on both 2L (the long arm of chromosome2) and 7L. The percentage distance from the centromere to the hybridization site was 61.8 for umc22 and 63.0 for umcl22 on 2L, and 47.5 and 48.9 on 7L respectively. It demonstrated that both umc22 and umcl22 had their homologous or homoeologous sequences on 7L besides 2L and they were duplicated sequences. Because genetic and physical maps show the same marker order, the gene ht1 should be physically located between the hybridization sites of umc22 and umcl22 on 2L and probably also should have its homologous or homoeologous sequence between the two hybridization sites on 7L. Actually, the signal spots showing the hybridization sites of the markers umcl22 and umc22 probably also represent the physical location of the ht1, because each signal spot could occupy about 7% of the arm, while the percentage distance between uncl22 and umc22 is only 1.2. The FISH techniques of small low copy DNA sequences for plants are discussed.

Cytogenetic mapping of disease resistance genes and analysis of their distribution features on chromosomes in maize

Cytogenetie maps of four clusters of disease resistance genes were generated by ISH of the two RFLP markers tightly linked to and flanking each of maize resistance genes and the cloned resistance genes from other plant species onto maize chromosomes, combining with data published before. These genes includeHelminthosporium turcium Pass resistance genes Ht1, Htn1 and Ht2,Helminthosporium maydis Nisik resistance genes Rhm1 and Rhm2, maize dwarf mosaic virus resistance gene Mdml, wheat streak mosaic virus resistance gene Wsm1,Helminthosporzum carbonum ULLstrup resistance gene Hm1 and the clonedXanthomonas oryzae pv.Oryzae resistance gene Xa21 of rice,Cladosporium fulvum resistance genes Cf-9 and Cf-2. 1 of tomato, andPseudomonas syringae resistance gene RPS2 ofArabidopsis. Most of the tested disease resistance genes located on the four chromosomes, i. e., chromosomes1, 3, 6 and 8, and they closely distributed at the interstitial regions of these chromosomal long arms with percentage distances ranging 31.44(±3.72)−72.40(±3.25) except for genes Rhm1, Rhm2, Mdm1 and Wsm1 which mapped on the satellites of the short arms of chromosome6. It showed that the tested RFLP markers and genes were duplicated or triplicated in maize genome. Homology and conservation of disease resistance genes among species, and relationship between distribution features and functions of the genes were discussed. The results provide Important scientific basis for deeply understanding structure and function of disease resistance genes and breeding in maize.

Predominance in New York of Isolates of Exserohilum turcicum Virulent on Maize with Gene Ht1

Predominance in New York of Isolates of Exserohilum turcicum Virulent on Maize with Gene Ht1

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3'-position. Similar to the flavonoid 3'-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3'-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3'-position in Petunia. Thus, the introduction of the 3'-hydroxyl group is clearly achieved by hydroxylation of C15-intermediates, and the concomitant occurrence of the 3',4'-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.

Modification of the B-ring during flavonoid synthesis in Petunia hybrida: Effect of the hydroxylation gene Hf1 on dihydroflavonol intermediates

The white flowering mutant W48 of Petunia hybrida is dominant for the hydroxylation gene Hf1 and homozygous recessive for the hydroxylation gene Ht1 and the anthocyanin gene An1. Flower buds of this mutant accumulate dihydrokaempferol-glucosides. Thus the effect of Hf1 being dominant is not the hydroxylation of the C15 skeleton, as is the case in mutants that are able to synthesize anthocyanins. This can be explained either by a feed-back inhibition of the hydroxylation by small amounts of dihydromyricetin (glucosides), or by a controlling effect of the gene An1 on the expression of Hf1. However, the white flowering mutant W58, which is homozygous recessive for the gene An6 and dominant for Hf1, accumulates dihydromyricetin (glucosides). This excludes a possible feed-back inhibition by dihydromyricetin and we conclude that An1 controls the expression of Hf1. Feeding of radioactive malonic acid to isolated flower limbs of mutants able to synthesize anthocyanins, leads to the incorporation of radioactivity into dihydrokaempferol (glucosides) and dihydroquercetin (glucosides). These results show that glucosylation of dihydroflavonols is a normal event in anthocyanin biosynthesis and is not induced by an inhibition of anthocyanin synthesis.

Modification of the B-ring during flavonoid synthesis in Petunia hybrida: Introduction of the 3?-hydroxyl group regulated by the gene Ht1

The flower-color mutants of Petunia hybrida W37 and W18, which are homozygous recessive for the anthocyanin gene An3, accumulate flavanone glycosides in the flowers. It is concluded that the gene An3 is not directly involved in the synthesis of the C15 skeleton, but that it probably takes part in modifying the skeleton. Complementation experiments with the mutants W18 and M5 show that the hydroxylating gene Ht1, which is reponsible for the introduction of the second hydroxyl group in the B-ring at position 3', is expressed after gene An3. In P. hybrida introduction of the 3'-hydroxyl group is therefore not achieved by specific incorporation of caffeic acid during synthesis of the C15 skeleton, but by hydroxylation of a C15 skeleton. When anthocyanin synthesis is blocked by homozygous recessive hydroxylating genes Ht1 and Hf1, as in the mutant M5, dihydrokaempferol-7-glucoside is accumulated. This intermediate is discussed as a possible substrate for B-ring hydroxylation.