Phytoplasmas such as "Candidatus Phytoplasma pruni," the causal agent of X-disease of stone fruits, lack detailed biological analysis. This has limited the understanding of plant resistance mechanisms. Chokecherry (Prunus virginiana L.) is a promising model to be used for the plant-phytoplasma interaction due to its documented ability to resist X-disease infection. A consensus chokecherry genetic map "Cho" was developed with JoinMap 4.0 by joining two parental maps. The new map contains a complete set of 16 linkage groups, spanning a genetic distance of 2,172 cM with an average marker density of 3.97 cM. Three significant quantitative trait loci (QTL) associated with X-disease resistance were identified contributing to a total of 45.9% of the phenotypic variation. This updated genetic linkage map and the identified QTL will provide the framework needed to facilitate molecular genetics, genomics, breeding, and biotechnology research concerning X-disease in chokecherry and other Prunus species.

In the context of climate warming, plants will be facing an increased risk of epidemics as well as the emergence of new highly aggressive pathogen species. Although a permanent increase of temperature strongly affects plant immunity, the underlying molecular mechanisms involved are still poorly characterized. In this study, we aimed to uncover the genetic bases of resistance mechanisms that are efficient at elevated temperature to the Ralstonia solanacearum species complex (RSSC), one of the most harmful phytobacteria causing bacterial wilt. To start the identification of quantitative trait loci (QTLs) associated with natural variation of response to R. solanacearum, we adopted a genome wide association (GWA) mapping approach using 176 worldwide natural accessions of Arabidopsis thaliana inoculated with the R. solanacearum GMI1000 strain. Following two different procedures of root-inoculation (root apparatus cut vs. uncut), plants were grown either at 27 or 30°C, with the latter temperature mimicking a permanent increase in temperature. At 27°C, the RPS4/RRS1-R locus was the main QTL of resistance detected regardless of the method of inoculation used. This highlights the power of GWA mapping to identify functionally important loci for resistance to the GMI1000 strain. At 30°C, although most of the accessions developed wilting symptoms, we identified several QTLs that were specific to the inoculation method used. We focused on a QTL region associated with response to the GMI1000 strain in the early stages of infection and, by adopting a reverse genetic approach, we functionally validated the involvement of a strictosidine synthase-like 4 (SSL4) protein that shares structural similarities with animal proteins known to play a role in animal immunity.

Plant growth-promoting bacteria (PGPB) are components of the plant rhizosphere that promote plant growth and/or inhibit pathogen activity. To explore the cotton seedlings response to Bacillus circulans GN03 with high efficiency of plant growth promotion and disease resistance, a pot experiment was carried out, in which inoculations levels of GN03 were set at 104 and 108 cfu⋅mL-1. The results showed that GN03 inoculation remarkably enhanced growth promotion as well as disease resistance of cotton seedlings. GN03 inoculation altered the microbiota in and around the plant roots, led to a significant accumulation of growth-related hormones (indole acetic acid, gibberellic acid, and brassinosteroid) and disease resistance-related hormones (salicylic acid and jasmonic acid) in cotton seedlings, as determined with ELISA, up-regulated the expression of phytohormone synthesis-related genes (EDS1, AOC1, BES1, and GA20ox), auxin transporter gene (Aux1), and disease-resistance genes (NPR1 and PR1). Comparative genomic analyses was performed between GN03 and four similar species, with regards to phenotype, biochemical characteristics, and gene function. This study provides valuable information for applying the PGPB alternative, GN03, as a plant growth and disease-resistance promoting fertilizer.

Fitness cost is a common phenomenon in rice blast disease-resistance breeding. MiR396 is a highly conserved microRNA (miRNA) family targeting Growth Regulating Factor (OsGRF) genes. Mutation at the target site of miR396 in certain OsGRF gene or blocking miR396 expression leads to increased grain yield. Here we demonstrated that fitness cost can be trade-off in miR396-OsGRFs module via balancing growth and immunity against the blast fungus. The accumulation of miR396 isoforms was significantly increased in a susceptible accession, but fluctuated in a resistant accession upon infection of Magnaporthe oryzae. The transgenic lines over-expressing different miR396 isoforms were highly susceptible to M. oryzae. In contrast, overexpressing target mimicry of miR396 to block its function led to enhanced resistance to M. oryzae in addition to improved yield traits. Moreover, transgenic plants overexpressing OsGRF6, OsGRF7, OsGRF8, and OsGRF9 exhibited enhanced resistance to M. oryzae, but showed different alteration of growth. While overexpression of OsGRF7 led to defects in growth, overexpression of OsGRF6, OsGRF8, and OsGRF9 resulted in better or no significant change of yield traits. Collectively, our results indicate that miR396 negatively regulates rice blast disease- resistance via suppressing multiple OsGRFs, which in turn differentially control growth and yield. Therefore, miR396-OsGRFs could be a potential module to demolish fitness cost in rice blast disease-resistance breeding.

Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented susceptibility conferred by the Arabidopsis ndr1-1 mutant to Pseudomonas syringae strains and also led to enhanced resistance to an oomycete pathogen Hyaloperonospora arabidopsidis. Such heightened resistance is associated with increased SA production and expression of the defense marker gene PATHOGENESIS RELATED 1 (PR1). In addition, we found that expression of PR1 and accumulation of SA were induced to modest levels in citrus infected with Candidatus Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that CsNDR1 is a functional ortholog of Arabidopsis NDR1. Since Ca. L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production.

One of the great challenges for food security in the 21st century is to improve yield stability through the development of disease-resistant crops. Crop research is often hindered by the lack of molecular tools, growth logistics, generation time and detailed genetic annotations, hence the power of model plant species. Our knowledge of plant immunity today has been largely shaped by the use of models, specifically through the use of mutants. We examine the importance of Arabidopsis and tomato as models in the study of plant immunity and how they help us in revealing a detailed and deep understanding of the various layers contributing to the immune system. Here we describe examples of how knowledge from models can be transferred to economically important crops resulting in new tools to enable and accelerate classical plant breeding. We will also discuss how models, and specifically transcriptomics and effectoromics approaches, have contributed to the identification of core components of the defense response which will be key to future engineering of durable and sustainable disease resistance in plants.

Disease resistance in plants is mostly quantitative, with both major and minor genes controlling resistance. This research aimed to optimize genomic selection (GS) models for use in breeding programs that are needed to select both major and minor genes for resistance. In this study, stripe rust (Puccinia striiformis Westend. f. sp. tritici Erikss.) of wheat (Triticum aestivum L.) was used as a model for quantitative disease resistance. The quantitative nature of stripe rust is usually phenotyped with two disease traits, infection type (IT) and disease severity (SEV). We compared two types of training populations composed of 2,630 breeding lines (BLs) phenotyped in single-plot trials from 4 years (2016-2020) and 475 diversity panel (DP) lines from 4 years (2013-2016), both across two locations. We also compared the accuracy of models using four different major gene markers and genome-wide association study (GWAS) markers as fixed effects. The prediction models used 31,975 markers that are replicated 50 times using a 5-fold cross-validation. We then compared GS models using a marker-assisted selection (MAS) to compare the prediction accuracy of the markers alone and in combination. GS models had higher accuracies than MAS and reached an accuracy of 0.72 for disease SEV. The major gene and GWAS markers had only a small to nil increase in the prediction accuracy more than the base GS model, with the highest accuracy increase of 0.03 for the major markers and 0.06 for the GWAS markers. There was a statistical increase in the accuracy using the disease SEV trait, BLs, population type, and combining years. There was also a statistical increase in the accuracy using the major markers in the validation sets as the mean accuracy decreased. The inclusion of fixed effects in low prediction scenarios increased the accuracy up to 0.06 for GS models using significant GWAS markers. Our results indicate that GS can accurately predict quantitative disease resistance in the presence of major and minor genes.

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum, Pythium irregulare, Pythium oopapillum, and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with "Williams," a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum, P. irregulare, and P. torulosum. Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare. These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens.

Nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain proteins are immune sensors and play critical roles in plant disease resistance. In this study, we cloned and characterized a novel NBS-LRR gene ZmNBS25 in maize. We found that ZmNBS25 could response to pathogen inoculation and salicylic acid (SA) treatment in maize, and transient overexpression of ZmNBS25 induced a hypersensitive response in tobacco. High-performance liquid chromatography (HPLC) analysis showed that, compared to control plants, ZmNBS25 overexpression (ZmNBS25-OE) in Arabidopsis and rice resulted in higher SA levels. By triggering the expression of certain defense-responsive genes, ZmNBS25-OE enhanced the resistance of Arabidopsis and rice to Pseudomonas syringae pv. tomato DC3000 and sheath blight disease, respectively. Moreover, we found little change of grain size and 1000-grain weight between ZmNBS25-OE rice lines and controls. Together, our results suggest that ZmNBS25 can function as a disease resistance gene across different species, being a valuable candidate for engineering resistance in breeding programs.

Karnal bunt (KB) of wheat, caused by Tilletia indica, is one of the greatest challenges to grain industry, not because of yield loss, but quarantine regulations that restrict international movement and trade of affected stocks. Genetic resistance is the best way to manage this disease. Although several different sources of resistance have been identified to date, very few of those have been subjected to genetic analyses. Understanding the genetics of resistance, characterization and mapping of new resistance loci can help in development of improved germplasm. The objective of this study was to identify and characterize resistance loci (QTL) in two independent recombinant inbred lines (RILs) populations utilizing different wheat lines as resistance donors. Elite CIMMYT wheat lines Blouk#1 and Huirivis#1 were used as susceptible female parents and WHEAR/KUKUNA/3/C80.1/3∗BATAVIA//2∗WBLL1 (WKCBW) and Mutus as moderately resistant male parents in Pop1 and Pop2 populations, respectively. Populations were evaluated for KB resistance in 2015-16 and 2016-17 cropping seasons at two seeding dates (total four environments) in Cd. Obregon, Mexico. Two stable QTL from each population were identified in each environment: QKb.cim-2B and QKb.cim-3D (Pop1), QKb.cim-3B1 and QKb.cim-5B2 (Pop2). Other than those four QTL, other QTL were detected in each population which were specific to environments: QKb.cim-5B1, QKb.cim-6A, and QKb.cim-7A (Pop1), QKb.cim-3B2, QKb.cim-4A1, QKb.cim-4A2, QKb.cim-4B, QKb.cim-5A1, QKb.cim-5A2, and QKb.cim-7A2 (Pop2). Among the four stable QTL, all but QKb.cim-3B1 were derived from the resistant parent. QKb.cim-2B and QKb.cim-3D in Pop1 and QKb.cim-3B1 and QKb.cim-5B2 in Pop2 explained 5.0-11.4% and 3.3-7.1% phenotypic variance, respectively. A combination of two stable QTL in each population reduced KB infection by 24-33%, respectively. Transgressive resistant segregants lines derived with resistance alleles from both parents in each population were identified. Single nucleotide polymorphism (SNP) markers flanking these QTL regions may be amenable to marker-assisted selection. The best lines from both populations (in agronomy, end-use quality and KB resistance) carrying resistance alleles at all identified loci, may be used for inter-crossing and selection of improved germplasm in future. Markers flanking these QTL may assist in selection of such lines.

Peanut diseases, such as leaf spot and spotted wilt caused by Tomato Spotted Wilt Virus, can significantly reduce yield and quality. Application of marker assisted plant breeding requires the development and validation of different types of DNA molecular markers. Nearly 10,000 SSR-based molecular markers have been identified by various research groups around the world, but less than 14.5% showed polymorphism in peanut and only 6.4% have been mapped. Low levels of polymorphism limit the application of marker assisted selection (MAS) in peanut breeding programs. Insertion/deletion (InDel) markers have been reported to be more polymorphic than SSRs in some crops. The goals of this study were to identify novel InDel markers and to evaluate the potential use in peanut breeding. Forty-eight InDel markers were developed from conserved sequences of functional genes and tested in a diverse panel of 118 accessions covering six botanical types of cultivated peanut, of which 104 were from the U.S. mini-core. Results showed that 16 InDel markers were polymorphic with polymorphic information content (PIC) among InDels ranged from 0.017 to 0.660. With respect to botanical types, PICs varied from 0.176 for fastigiata var., 0.181 for hypogaea var., 0.306 for vulgaris var., 0.534 for aequatoriana var., 0.556 for peruviana var., to 0.660 for hirsuta var., implying that aequatoriana var., peruviana var., and hirsuta var. have higher genetic diversity than the other types and provide a basis for gene functional studies. Single marker analysis was conducted to associate specific marker to disease resistant traits. Five InDels from functional genes were identified to be significantly correlated to tomato spotted wilt virus (TSWV) infection and leaf spot, and these novel markers will be utilized to identify disease resistant genotype in breeding populations.

Immune responses in plants can be triggered by damage/microbe-associated molecular patterns (DAMPs/MAMPs) upon recognition by plant pattern recognition receptors (PRRs). DAMPs are signaling molecules synthesized by plants or released from host cellular structures (e.g., plant cell walls) upon pathogen infection or wounding. Despite the hypothesized important role of plant cell wall-derived DAMPs in plant-pathogen interactions, a very limited number of these DAMPs are well characterized. Recent work demonstrated that pectin-enriched cell wall fractions extracted from the cell wall mutant impaired in Arabidopsis Response Regulator 6 (arr6), that showed altered disease resistance to several pathogens, triggered more intense immune responses than those activated by similar cell wall fractions from wild-type plants. It was hypothesized that arr6 cell wall fractions could be differentially enriched in DAMPs. In this work, we describe the characterization of the previous immune-active fractions of arr6 showing the highest triggering capacities upon further fractionation by chromatographic means. These analyses pointed to a role of pentose-based oligosaccharides triggering plant immune responses. The characterization of several pentose-based oligosaccharide structures revealed that β-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance.

Intercropping could alleviate soil-borne diseases, however, few studies focused on the immunity of the host plant induced by the interspecific interactions. To test whether or not intercropping could enhance the disease resistance of host plant, we investigated the effect of companion cropping with potato onion on tomato Verticillium wilt caused by Verticillium dahliae (V. dahliae). To investigate the mechanisms, the root exudates were collected from tomato and potato onion which were grown together or separately, and were used to examine the antifungal activities against V. dahliae in vitro, respectively. Furthermore, RNA-seq was used to examine the expression pattern of genes related to disease resistance in tomato companied with potato onion compared to that in tomato grown alone, under the condition of infection with V. dahliae. The results showed that companion cropping with potato onion could alleviate the incidence and severity of tomato Verticillium wilt. The further studies revealed that the root exudates from tomato companied with potato onion significantly inhibited the mycelia growth and spore germination of V. dahliae. However, there were no significant effects on these two measurements for the root exudates from potato onion grown alone or from potato onion grown with tomato. RNA-seq data analysis showed the disease defense genes associated with pathogenesis-related proteins, biosynthesis of lignin, hormone metabolism and signal transduction were expressed much higher in the tomato companied with potato onion than those in the tomato grown alone, which indicated that these defense genes play important roles in tomato against V. dahliae infection, and meant that the disease resistance of tomato against V. dahliae was enhanced in the companion copping with potato onion. We proposed that companion cropping with potato onion could enhance the disease resistance of tomato against V. dahliae by regulating the expression of genes related to disease resistance response. This may be a potential mechanism for the management of soil-borne plant diseases in the intercropping system.

Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD.

The interaction of roses with the leaf spot pathogen Diplocarpon rosae (the cause of black spot on roses) is an interesting pathosystem because it involves a long-lived woody perennial, with life history traits very different from most model plants, and a hemibiotrophic pathogen with moderate levels of gene flow. Here we present data on the molecular structure of the first monogenic dominant resistance gene from roses, Rdr1, directed against one isolate of D. rosae. Complete sequencing of the locus carrying the Rdr1 gene resulted in a sequence of 265,477 bp with a cluster of nine highly related TIR-NBS-LRR (TNL) candidate genes. After sequencing revealed candidate genes for Rdr1, we implemented a gene expression analysis and selected five genes out of the nine TNLs. We then silenced the whole TNL gene family using RNAi (Rdr1-RNAi) constructed from the most conserved sequence region and demonstrated a loss of resistance in the normally resistant genotype. To identify the functional TNL gene, we further screened the five TNL candidate genes with a transient leaf infiltration assay. The transient expression assay indicated a single TNL gene (muRdr1H), partially restoring resistance in the susceptible genotype. Rdr1 was found to localize within the muRdr1 gene family; the genes within this locus contain characteristic motifs of active TNL genes and belong to a young cluster of R genes. The transient leaf assay can be used to further analyze the rose black spot interaction and its evolution, extending the analyses to additional R genes and to additional pathogenic types of the pathogen.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

Nitric oxide (NO) is a signaling molecule that regulates various processes, including plant growth and development, immunity, and environmental interactions. Using high throughput RNA-seq data, we explored the role of the NO-induced ATILL6 gene in plant growth and defense using functional genomics. The atill6 mutant and wild-types were challenged with either oxidative (H2O2, MV) or nitro-oxidative (CySNO, GSNO) stress conditions, and the phenotypic results showed that ATILL6 gene differentially regulates cotyledon development frequency (CDF) as well as the root and shoot lengths of the plants. To investigate whether ATILL6 plays a role in plant basal or resistance (R)-gene-mediated defense, the plants were challenged with either virulent or avirulent strains of Pseudomonas syringae pathovar tomato (Pst) DC3000. The atill6 line showed a susceptible phenotype, higher pathogen growth, and highly reduced transcript accumulation of PR1 and PR2 genes. These results suggested that ATILL6 positively regulates plant basal defense. Furthermore, after the inoculation of atill6 with avirulent Pst (DC3000), the expressions of the PR1 and PR2 genes decreased, suggesting a positive role in R-gene-mediated resistance in protecting the plant from further spread of disease. We also investigated the role of ATILL6 in systemic acquired resistance (SAR), and the results showed that ATILL6 positively regulates SAR, as the mutant line atill6 has significantly (p ≤ 0.05) lower transcript accumulation of PR, G3DPH, and AZI genes. Overall, these results indicate that the NO-induced ATILL6 gene differentially regulates plant growth and positively regulates plant basal defense, R-gene-mediated resistance, and SAR.

Verticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in multiple strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar "Redgauntlet" and subclade II-2 specific resistance in "Fenella" and "Chandler." A large-scale low-cost phenotyping platform was developed utilizing automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores. A co-dominant resistant QTL; FaRVd3D, present in both "Redgauntlet" and "Hapil" cultivars exhibited a major effect of 18.3% when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the "Emily" cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the F1 mapping population. Markers identified in populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from "Redgauntlet" and FaRVd6D from "Chandler" were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment.

Over the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an in planta expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals in planta, plants can be protected from pathogens, and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed vs. manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. Since no HLB-resistant citrus cultivars have been identified, we attempted to develop citrus resistant to both HLB and citrus canker through overexpression of a modified plant thionin. To improve effectiveness for disease resistance, we modified and synthesized the sequence encoding a plant thionin and cloned into the binary vector pBinPlus/ARS. The construct was then introduced into Agrobacterium strain EHA105 for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were generated by Agrobacterium-mediated transformation. Successful transformation and transgene gene expression was confirmed by molecular analysis. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at a range of concentrations, and a significant reduction in canker symptoms and a decrease in bacterial growth were demonstrated compared to nontransgenic plants. Furthermore, the transgenic citrus plants were challenged with HLB via graft inoculation. Our results showed significant Las titer reduction in roots of transgenic Carrizo compared with control plants and reduced scion Las titer 12 months after graft inoculation. These data provide promise for engineering citrus disease resistance against HLB and canker.