On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

Genomic regions associated with virulence in Setosphaeria turcica identified by linkage mapping in a biparental population.

Northern corn leaf blight (NCLB) and sorghum leaf blight (SLB) are significant diseases of maize and sorghum, respectively, caused by the filamentous fungus Setosphaeria turcica. Strains of S. turcica are typically host-specific and infect either maize or sorghum. Host specificity in this pathogen is attributed to a single locus for maize and a second distinct locus for sorghum. To identify the genetic basis of host specificity in S. turcica, we generated a biparental population of S. turcica by crossing strains specific to maize and sorghum, phenotyped the population for leaf blight on sorghum and maize, genotyped the population to create a linkage map of S. turcica, and located candidate virulence regions. A total of 190 ascospores from 35 pseudothecia were isolated from the cross of maize and sorghum-specific strains. Greenhouse phenotyping of the biparental population (n = 144) showed independent inheritance of virulence, as indicated by a 1:1:1:1 segregation for virulence to maize, sorghum, both maize and sorghum, and avirulence to both crops. The population and host-specific parent strains were genotyped using genome skim sequencing on an Illumina NovaSeq 6000 platform resulting in over 780 million reads. A total of 32,635 variants including single nucleotide polymorphisms and indels were scored. There was evidence for a large deletion in the sorghum-specific strain of S. turcica. A genetic map consisting of 17 linkage groups spanning 3,069 centimorgans was constructed. Virulence to sorghum and maize mapped on distinct linkage groups with a significant QTL detected for virulence to maize. Furthermore, a single locus each for the in vitro traits hyphal growth rate and conidiation were identified and mapped onto two other linkage groups. In vitro traits did not correlate with in planta virulence complexity, suggesting that virulence on both hosts does not incur a fitness cost. Hyphal growth rate and conidiation were negatively correlated, indicating differences in hyphal growth versus dispersal ability for this pathogen. Identification of genetic regions underlying virulence specificity and saprotrophic growth traits in S. turcica provides a better understanding of the S. turcica- Andropogoneae pathosystem.

Conserved defense responses between maize and sorghum to Exserohilum turcicum.

BACKGROUND: Exserohilum turcicum is an important pathogen of both sorghum and maize, causing sorghum leaf blight and northern corn leaf blight. Because the same pathogen can infect and cause major losses for two of the most important grain crops, it is an ideal pathosystem to study plant-pathogen evolution and investigate shared resistance mechanisms between the two plant species. To identify sorghum genes involved in the E. turcicum response, we conducted a genome-wide association study (GWAS). RESULTS: Using the sorghum conversion panel evaluated across three environments, we identified a total of 216 significant markers. Based on physical linkage with the significant markers, we detected a total of 113 unique candidate genes, some with known roles in plant defense. Also, we compared maize genes known to play a role in resistance to E. turcicum with the association mapping results and found evidence of genes conferring resistance in both crops, providing evidence of shared resistance between maize and sorghum. CONCLUSIONS: Using a genetics approach, we identified shared genetic regions conferring resistance to E. turcicum in both maize and sorghum. We identified several promising candidate genes for resistance to leaf blight in sorghum, including genes related to R-gene mediated resistance. We present significant advancements in the understanding of host resistance to E. turcicum, which is crucial to reduce losses due to this important pathogen.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Advances and Challenges in Bacterial Spot Resistance Breeding in Tomato (Solanum lycopersicum L.).

Bacterial spot is a serious disease of tomato caused by at least four species of Xanthomonas. These include X. euvesicatoria (race T1), X. vesicatoria (race T2), X. perforans (races T3 and T4), and X. gardneri, with the distinct geographical distribution of each group. Currently, X. gardneri and X. perforans are two major bacterial pathogens of tomato in North America, with X. perforans (race T4) dominating in east-coast while X. gardneri dominating in the Midwest. The disease causes up to 66% yield loss. Management of this disease is challenging due to the lack of useful chemical control measures and commercial resistant cultivars. Although major genes for resistance (R) and quantitative resistance have been identified, breeding tomato for resistance to bacterial spot has been impeded by multiple factors including the emergence of new races of the pathogen that overcome the resistance, multigenic control of the resistance, linkage drag, non-additive components of the resistance and a low correlation between seedling assays and field resistance. Transgenic tomato with Bs2 and EFR genes was effective against multiple races of Xanthomonas. However, it has not been commercialized because of public concerns and complex regulatory processes. The genomics-assisted breeding, effectors-based genomics breeding, and genome editing technology could be novel approaches to achieve durable resistance to bacterial spot in tomato. The main goal of this paper is to understand the current status of bacterial spot of tomato including its distribution and pathogen diversity, challenges in disease management, disease resistance sources, resistance genetics and breeding, and future prospectives with novel breeding approaches.

Phenotypic and Genetic Diversity of Xanthomonas perforans Populations from Tomato in North Carolina.

Bacterial spot caused by Xanthomonas spp. is one of the most devastating diseases of tomato in North Carolina (NC). In total, 290 strains of Xanthomonas spp. from tomato in NC collected over 2 years (2015 and 2016) were analyzed for phenotypic and genetic diversity. In vitro copper and streptomycin sensitivity assays revealed that >95% (n = 290) of the strains were copper tolerant in both years, whereas 25% (n = 127) and 46% (n = 163) were streptomycin tolerant in 2016 and 2015, respectively. Using BOX repetitive element PCR assay, fingerprint patterns showed four haplotypes (H1, H2, H3, and H4) among the strains analyzed. The multiplex real-time quantitative PCR on a subset of representative strains (n = 45) targeting the highly conserved hrcN gene identified Xanthomonas strains from tomato in NC that belonged to X. perforans. Race profiling of the representative strains (n = 45) on tomato and pepper differentials confirmed that ∼9 and 91% of strains are tomato races T3 and T4, respectively. Additionally, PCR assays and sequence alignments confirmed that the copL, copA, copB (copLAB copper tolerance gene cluster), and avrXv4 genes are present in the strains analyzed. Phylogenetic and comparative sequence analyses of six genomic regions (elongation factor G [fusA], glyceraldehyde-3-phosphate dehydrogenase A [gapA], citrate synthase [gltA], gyrase subunit B [gyrB], ABC transporter sugar permease [lacF], and GTP binding protein [lepA]) suggested that 13 and 74% of X. perforans strains from NC were genetically similar to races T3 and T4 from Florida, respectively. Our results provide insights that bacterial spot management practices in tomato should focus on deploying resistance genes to combat emerging pathogenic races of X. perforans and overcome the challenges currently posed by intense use of copper-based bactericides.

Current Status of Early Blight Resistance in Tomato: An Update.

Early blight (EB) is one of the dreadful diseases of tomato caused by several species of Alternaria including Alternaria linariae (which includes A. solani and A. tomatophila), as well as A. alternata. In some instances, annual economic yield losses due to EB have been estimated at 79%. Alternaria are known only to reproduce asexually, but a highly-virulent isolate has the potential to overcome existing resistance genes. Currently, cultural practices and fungicide applications are employed for the management of EB due to the lack of strong resistant cultivars. Resistance sources have been identified in wild species of tomato; some breeding lines and cultivars with moderate resistance have been developed through conventional breeding methods. Polygenic inheritance of EB resistance, insufficient resistance in cultivated species and the association of EB resistance with undesirable horticultural traits have thwarted the effective breeding of EB resistance in tomato. Several quantitative trait loci (QTL) conferring EB resistance have been detected in the populations derived from different wild species including Solanum habrochaites, Solanum arcanum and S. pimpinellifolium, but none of them could be used in EB resistance breeding due to low individual QTL effects. Pyramiding of those QTLs would provide strong resistance. More research is needed to identify additional sources of useful resistance, to incorporate resistant QTLs into breeding lines through marker-assisted selection (MAS) and to develop resistant cultivars with desirable horticultural traits including high yielding potential and early maturity. This paper will review the current understanding of causal agents of EB of tomato, resistance genetics and breeding, problems associated with breeding and future prospects.

Identification of loci conferring resistance to 4 foliar diseases of maize.

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.

Identification of quantitative trait loci associated with bacterial spot race T4 resistance in intra-specific populations of tomato (Solanum lycopersicum L.).

Bacterial spot of tomato is a serious disease caused by at least four species and four races of Xanthomonas- X. euvesicatoria (race T1), X. vesicatoria (race T2), X. perforans (race T3 and T4), and X. gardneri, with X. perforans race T4 being predominant in the southeast USA. Practical management of this disease is challenging because of the need for more effective chemicals and commercially resistant cultivars. Identification of genetic resistance is the first step to developing a disease-resistant variety. The objective of this study was to identify quantitative trait loci (QTL) conferring resistance to race T4 in two independent recombinant inbred lines (RILs) populations NC 10204 (intra-specific) and NC 13666 (interspecific) developed by crossing NC 30P x NC22L-1(2008) and NC 1CELBR x PI 270443, respectively. Seven QTLs on chromosomes 2, 6, 7, 11, and 12 were identified in NC 10204. The QTL on chromosome 6 explained the highest percentage of phenotypic variance (up to 21.3%), followed by the QTL on chromosome 12 (up to 8.2%). On the other hand, the QTLs on chromosomes 1, 3, 4, 6, 7, 8, 9, and 11 were detected in NC 13666. The QTLs on chromosomes 6, 7, and 11 were co-located in NC 10204 and NC 13666 populations. The donor of the resistance associated with these QTL in NC 10204 is a released breeding line with superior horticultural traits. Therefore, both the donor parent and the QTL information will be useful in tomato breeding programs as there will be minimal linkage drag associated with the bacterial spot resistance.

Genetic mapping of sorghum resistance to an Illinois isolate of Colletotrichum sublineola.

Anthracnose leaf blight (ALB) is an economically important disease of sorghum [Sorghum bicolor (L.) Moench] caused by the fungal pathogen Colletotrichum sublineola Henn. ex Sacc. & Trotter. Although qualitative and quantitative resistance have been identified for ALB, the usefulness of resistance loci differs depending on the pathogen pathotype. Identifying resistance effective against unique pathogen pathotypes is critical to managing ALB, as the disease is managed primarily through the deployment of host resistance. We isolated C. sublineola from ALB-infected leaves collected in Illinois and found that the strain was a novel pathotype, as it produced a unique combination of virulence against a set of differential lines. Using this isolate, we inoculated 579 temperate-adapted sorghum conversion lines in 2019 and 2020. We then conducted a genome-wide association study (GWAS) and a metabolic pathway analysis using the Pathway Associated Study Tool (PAST). We identified 47 significant markers distributed across all chromosomes except chromosome 8. We identified 32 candidate genes based on physical proximity with significant markers, some of which have a known role in host defense. We identified 47 pathways associated with ALB resistance, indicating a role for secondary metabolism in defense to ALB. Our results are important to improve the understanding of the genetic basis of ALB resistance in sorghum and highlight the importance of developing durable resistance to ALB.