The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 ...(PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On).
To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants.
CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.
Recent studies on plant immunity have suggested that a pathogen should suppress induced plant defense in order to infect a plant species, which otherwise would have been a nonhost to the pathogen. ...For this purpose, pathogens exploit effector molecules to interfere with different layers of plant defense responses. In this review, we summarize the latest findings on plant factors that are activated by pathogen effectors to suppress plant immunity. By looking from a different point of view into host and nonhost resistance, we propose a novel breeding strategy: disabling plant disease susceptibility genes (S-genes) to achieve durable and broad-spectrum resistance.
Potato defends against Phytophthora infestans infection by resistance (R)-gene-based qualitative resistance as well as a quantitative field resistance. R genes are renowned to be rapidly overcome by ...this oomycete, and potato cultivars with a decent and durable resistance to current P. infestans populations are hardly available. However, potato cultivar Sarpo Mira has retained resistance in the field over several years. We dissected the resistance of 'Sarpo Mira' in a segregating population by matching the responses to P. infestans RXLR effectors with race-specific resistance to differential strains. The resistance is based on the combination of four pyramided qualitative R genes and a quantitative R gene that was associated with field resistance. The qualitative R genes include R3a, R3b, R4, and the newly identified Rpi-Smira1. The qualitative resistances matched responses to avirulence (AVR)3a, AVR3b, AVR4, and AVRSmira1 RXLR effectors and were overcome by particular P. infestans strains. The quantitative resistance was determined to be conferred by a novel gene, Rpi-Smira2. It was only detected under field conditions and was associated with responses to the RXLR effector AvrSmira2. We foresee that effector-based resistance breeding will facilitate selecting and combining qualitative and quantitative resistances that may lead to a more durable resistance to late blight.
Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum ...disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.
Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security(1,2). All late blight resistance genes identified to date belong ...to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors(3). However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors(4). Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.
Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and ...the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala' was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana'.
Phytophthora infestans
is the causal agent of late blight in potato. The Mexican species
Solanum demissum
is well known as a good resistance source. Among the 11
R
gene differentials, which were ...introgressed from
S. demissum
, especially
R8
and
R9
differentials showed broad spectrum resistance both under laboratory and under field conditions. In order to gather more information about the resistance of the
R8
and
R9
differentials, F1 and BC1 populations were made by crossing Mastenbroek (Ma)
R8
and
R9
clones to susceptible plants. Parents and offspring plants were examined for their pathogen recognition specificities using agroinfiltration with known
Avr
genes, detached leaf assays (DLA) with selected isolates, and gene-specific markers. An important observation was the discrepancy between DLA and field trial results for
Pi
isolate IPO-C in all F1 and BC1 populations, so therefore also field trial results were included in our characterization. It was shown that in Ma
R8
and Ma
R9
, respectively, at least four (
R3a
,
R3b
,
R4,
and
R8
) and seven (
R1
,
Rpi-abpt1
,
R3a
,
R3b
,
R4
,
R8
,
R9
)
R
genes were present. Analysis of Ma
R8
and Ma
R9
offspring plants, that contained different combinations of multiple resistance genes, showed that
R
gene stacking contributed to the
Pi
recognition spectrum. Also, using a
Pi
virulence monitoring system in the field, it was shown that stacking of multiple
R
genes strongly delayed the onset of late blight symptoms. The contribution of
R8
to this delay was remarkable since a plant that contained only the
R8
resistance gene still conferred a delay similar to plants with multiple resistance genes, like, e.g., cv Sarpo Mira. Using this “de-stacking” approach, many
R
gene combinations can be made and tested in order to select broad spectrum
R
gene stacks that potentially provide enhanced durability for future application in new late blight resistant varieties.
Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are ...conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in
species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species
subsp.
. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (
) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of
species.
Apple (Malus×domesticaBorkh) is among the main sources of phenolic compounds in the human diet. The genetic basis of the quantitative variations of these potentially beneficial phenolic compounds was ...investigated. A segregating F₁ population was used to map metabolite quantitative trait loci (mQTLs). Untargeted metabolic profiling of peel and flesh tissues of ripe fruits was performed using liquid chromatography–mass spectrometry (LC-MS), resulting in the detection of 418 metabolites in peel and 254 in flesh. In mQTL mapping using MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 in the flesh. Four linkage groups (LGs), LG1, LG8, LG13, and LG16, were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. The genetics of annotated metabolites was studied in more detail using MapQTL®. A number of quercetin conjugates had mQTLs on LG1 or LG13. The most important mQTL hotspot with the largest number of metabolites was detected on LG16: mQTLs for 33 peel-related and 17 flesh-related phenolic compounds. Structural genes involved in the phenylpropanoid biosynthetic pathway were located, using the apple genome sequence. The structural geneleucoanthocyanidin reductase(LAR1) was in the mQTL hotspot on LG16, as were seven transcription factor genes. The authors believe that this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.
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