Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Identification and Mapping of bs8, a Novel Locus Conferring Resistance to Bacterial Spot Caused by Xanthomonas gardneri.

Although cultivars possessing recessive resistance alleles provide effective control of bacterial spot of pepper (Capsicum annuum), the deployed resistance gene, bs5, is ineffective against Xanthomonas gardneri, one of the pathogenic species. Resistance against X. gardneri was identified in C. annuum accession PI 163192, and this study sought to characterize this novel resistance and to map the resistance gene(s) to the pepper genome. We crossed PI 163192 with the susceptible cultivar Early Calwonder (ECW) to develop resistant near-isogenic lines (NILs) of ECW, designated ECW80R. The novel resistance in ECW80R was determined to be quantitative, recessively inherited, and non-hypersensitive-response causing, and inhibits lesion expansion and chlorosis. Presence of the resistance in NILs decreased the in planta bacterial population by ninefold compared with ECW. Bulked segregant analysis of resistant and susceptible individuals from an F2 population using whole genome single nucleotide polymorphisms identified a major resistance locus within an approximate 6-Mbp interval on the subtelomeric region of chromosome 11. We developed markers spanning this region and used these to genotype backcross F2 populations, which further delimited the resistance locus within a 2.3-Mbp interval. The novel resistance locus has been designated bs8. ECW80R and the linked markers developed in this study should prove useful for breeders seeking to advance this resistance into commercially relevant germplasm and for pyramiding bs8 with other resistance alleles such as bs5 and bs6. The allele bs8 will help prolong the durability of bacterial spot resistance in pepper and improve resistance to multiple species of Xanthomonas.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

Genome-wide analysis of flanking sequences reveals that Tnt1 insertion is positively correlated with gene methylation in Medicago truncatula.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

Transcriptome-based analyses of phosphite-mediated suppression of rust pathogens Puccinia emaculata and Phakopsora pachyrhizi and functional characterization of selected fungal target genes.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Draft Genome Sequence Resource of Switchgrass Rust Pathogen, Puccinia novopanici Isolate Ard-01.

Puccinia novopanici is an important biotrophic fungal pathogen that causes rust disease in switchgrass. Lack of genomic resources for P. novopanici has hampered the progress toward developing effective disease resistance against this pathogen. Therefore, we have sequenced the whole genome of P. novopanici and generated a framework to understand pathogenicity mechanisms and identify effectors, repeat element invasion, genome evolution, and comparative genomics among Puccinia spp. in the future. Long- and short-read sequences were generated from P. novopanici genomic DNA by PacBio and Illumina technologies, respectively, and assembled a 99.9-Mb genome. Transcripts of P. novopanici were predicted from assembled genome using MAKER and were further validated by RNAseq data. The genome sequence information of P. novopanici will be a valuable resource for researchers working on monocot rusts and plant disease resistance in general.

Metabolic flux towards the (iso)flavonoid pathway in lignin modified alfalfa lines induces resistance against Fusarium oxysporum f. sp. medicaginis.

Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or shift/spillover of flux towards the (iso)flavonoid pathway in CCoAOMT downregulated plants is associated with induced disease resistance.

Characterization of Brachypodium distachyon as a nonhost model against switchgrass rust pathogen Puccinia emaculata.

BACKGROUND: Switchgrass rust, caused by Puccinia emaculata, is an important disease of switchgrass, a potential biofuel crop in the United States. In severe cases, switchgrass rust has the potential to significantly affect biomass yield. In an effort to identify novel sources of resistance against switchgrass rust, we explored nonhost resistance against P. emaculata by characterizing its interactions with six monocot nonhost plant species. We also studied the genetic variations for resistance among Brachypodium inbred accessions and the involvement of various defense pathways in nonhost resistance of Brachypodium. RESULTS: We characterized P. emaculata interactions with six monocot nonhost species and identified Brachypodium distachyon (Bd21) as a suitable nonhost model to study switchgrass rust. Interestingly, screening of Brachypodium accessions identified natural variations in resistance to switchgrass rust. Brachypodium inbred accessions Bd3-1 and Bd30-1 were identified as most and least resistant to switchgrass rust, respectively, when compared to tested accessions. Transcript profiling of defense-related genes indicated that the genes which were induced in Bd21after P. emaculata inoculation also had higher basal transcript abundance in Bd3-1 when compared to Bd30-1 and Bd21 indicating their potential involvement in nonhost resistance against switchgrass rust. CONCLUSION: In the present study, we identified Brachypodium as a suitable nonhost model to study switchgrass rust which exhibit type I nonhost resistance. Variations in resistance response were also observed among tested Brachypodium accessions. Brachypodium nonhost resistance against P. emaculata may involve various defense pathways as indicated by transcript profiling of defense related genes. Overall, this study provides a new avenue to utilize novel sources of nonhost resistance in Brachypodium against switchgrass rust.

Host versus nonhost resistance: distinct wars with similar arsenals.

Plants face several challenges by bacterial, fungal, oomycete, and viral pathogens during their life cycle. In order to defend against these biotic stresses, plants possess a dynamic, innate, natural immune system that efficiently detects potential pathogens and initiates a resistance response in the form of basal resistance and/or resistance (R)-gene-mediated defense, which is often associated with a hypersensitive response. Depending upon the nature of plant-pathogen interactions, plants generally have two main defense mechanisms, host resistance and nonhost resistance. Host resistance is generally controlled by single R genes and less durable compared with nonhost resistance. In contrast, nonhost resistance is believed to be a multi-gene trait and more durable. In this review, we describe the mechanisms of host and nonhost resistance against fungal and bacterial plant pathogens. In addition, we also attempt to compare host and nonhost resistance responses to identify similarities and differences, and their practical applications in crop improvement.

Mapping of the bs5 and bs6 non-race-specific recessive resistances against bacterial spot of pepper.

Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregating populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6. We identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. This research enhances marker-assisted selection of bs5 and bs6 in breeding programs and is a crucial step towards elucidating the molecular mechanisms underlying the resistances.

Barley Stripe Mosaic Virus (BSMV)-Based Virus-Induced Gene Silencing to Functionally Characterize Genes in Wheat and Barley.

Virus-induced gene silencing (VIGS) is an efficient method for functional characterization of genes in monocot and dicot plants via transient silencing of gene(s) of interest. Among various virus vectors, Barley stripe mosaic virus (BSMV) is established as a vector of choice to silence genes in wheat and barley. BSMV is a single-stranded positive-sense RNA virus with a tripartite genome consisting of α, ß, and γ RNAs. BSMV-based VIGS has been used to silence both abiotic and biotic stress response genes in various growth stages of plants. Here we describe an efficient and effective protocol to successfully silence wheat and barley genes expressing in various tissues using this approach.

Comparative Genome Analyses of Plant Rust Pathogen Genomes Reveal a Confluence of Pathogenicity Factors to Quell Host Plant Defense Responses.

Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21-0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen-Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.

Genome-wide association analysis permits characterization of Stagonospora nodorum blotch (SNB) resistance in hard winter wheat.

Stagonospora nodorum blotch (SNB) is an economically important wheat disease caused by the necrotrophic fungus Parastagonospora nodorum. SNB resistance in wheat is controlled by several quantitative trait loci (QTLs). Thus, identifying novel resistance/susceptibility QTLs is crucial for continuous improvement of the SNB resistance. Here, the hard winter wheat association mapping panel (HWWAMP) comprising accessions from breeding programs in the Great Plains region of the US, was evaluated for SNB resistance and necrotrophic effectors (NEs) sensitivity at the seedling stage. A genome-wide association study (GWAS) was performed to identify single-nucleotide polymorphism (SNP) markers associated with SNB resistance and effectors sensitivity. We found seven significant associations for SNB resistance/susceptibility distributed over chromosomes 1B, 2AL, 2DS, 4AL, 5BL, 6BS, and 7AL. Two new QTLs for SNB resistance/susceptibility at the seedling stage were identified on chromosomes 6BS and 7AL, whereas five QTLs previously reported in diverse germplasms were validated. Allele stacking analysis at seven QTLs explained the additive and complex nature of SNB resistance. We identified accessions ('Pioneer-2180' and 'Shocker') with favorable alleles at five of the seven identified loci, exhibiting a high level of resistance against SNB. Further, GWAS for sensitivity to NEs uncovered significant associations for SnToxA and SnTox3, co-locating with previously identified host sensitivity genes (Tsn1 and Snn3). Candidate region analysis for SNB resistance revealed 35 genes of putative interest with plant defense response-related functions. The QTLs identified and validated in this study could be easily employed in breeding programs using the associated markers to enhance the SNB resistance in hard winter wheat.

The Immune Receptor Roq1 Confers Resistance to the Bacterial Pathogens Xanthomonas, Pseudomonas syringae, and Ralstonia in Tomato.

Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide-binding, leucine-rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.

Exploring natural variation for rice sheath blight resistance in Brachypodium distachyon.

Sheath blight caused by the soil borne fungus Rhizoctonia solani AG1-IA is one of the major diseases of rice in the world. Genetic resistance in rice against this disease has not been very successful. Brachypodium distachyon is considered as a model species for several cereal crops and it has been studied in the past to identify novel sources of disease resistance against cereal crop diseases. Therefore, the current study was designed to explore nonhost disease resistance in Brachypodium accessions against sheath blight pathogen of rice, Rhizoctonia solani. A total of 19 Brachypodium distachyon accessions were screened for resistance against Rhizoctonia solani AG1-IA. Different levels of resistance reactions were observed among accessions. Quantification of jasmonic acid (JA) and salicylic acid (SA) concentration in selected resistant (Bd3-1), moderately susceptible (Bd21), and susceptible (Bd30-1) inbred accessions revealed that Bd3-1 accumulated more JA upon pathogen infection compared to Bd21 or Bd30-1. In contrary, no differences were observed for SA accumulation in tested accessions suggesting that the resistance to R. solani in Brachypodium is due to an SA-independent defense pathway. Our study provides a new foundation to explore this area for more durable resistance against this disease.

Tissue Culture (Somatic Embryogenesis)-Induced Tnt1 Retrotransposon-Based Mutagenesis in Brachypodium distachyon.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.

Transcriptomic and metabolomic analyses identify a role for chlorophyll catabolism and phytoalexin during Medicago nonhost resistance against Asian soybean rust.

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a devastating foliar disease affecting soybean production worldwide. Understanding nonhost resistance against ASR may provide an avenue to engineer soybean to confer durable resistance against ASR. We characterized a Medicago truncatula-ASR pathosystem to study molecular mechanisms of nonhost resistance. Although urediniospores formed appressoria and penetrated into epidermal cells of M. truncatula, P. pachyrhizi failed to sporulate. Transcriptomic analysis revealed the induction of phenylpropanoid, flavonoid and isoflavonoid metabolic pathway genes involved in the production of phytoalexin medicarpin in M. truncatula upon infection with P. pachyrhizi. Furthermore, genes involved in chlorophyll catabolism were induced during nonhost resistance. We further characterized one of the chlorophyll catabolism genes, Stay-green (SGR), and demonstrated that the M. truncatula sgr mutant and alfalfa SGR-RNAi lines showed hypersensitive-response-like enhanced cell death upon inoculation with P. pachyrhizi. Consistent with transcriptomic analysis, metabolomic analysis also revealed the accumulation of medicarpin and its intermediate metabolites. In vitro assay showed that medicarpin inhibited urediniospore germination and differentiation. In addition, several triterpenoid saponin glycosides accumulated in M. truncatula upon inoculation with P. pachyrhizi. In summary, using multi-omic approaches, we identified a correlation between phytoalexin production and M. truncatula defense responses against ASR.