Transcriptomics of temperature-sensitive R gene-mediated resistance identifies a WAKL10 protein interaction network.

Understanding temperature-sensitivity of R gene-mediated resistance against apoplastic pathogens is important for sustainable food production in the face of global warming. Here, we show that resistance of Brassica napus cotyledons against Leptosphaeria maculans was temperature-sensitive in introgression line Topas-Rlm7 but temperature-resilient in Topas-Rlm4. A set of 1,646 host genes was differentially expressed in Topas-Rlm4 and Topas-Rlm7 in response to temperature. Amongst these were three WAKL10 genes, including BnaA07g20220D, representing the temperature-sensitive Rlm7-1 allele and Rlm4. Network analysis identified a WAKL10 protein interaction cluster specifically for Topas-Rlm7 at 25 °C. Diffusion analysis of the Topas-Rlm4 network identified WRKY22 as a putative regulatory target of the ESCRT-III complex-associated protein VPS60.1, which belongs to the WAKL10 protein interaction community. Combined enrichment analysis of gene ontology terms considering gene expression and network data linked vesicle-mediated transport to defence. Thus, dysregulation of effector-triggered defence in Topas-Rlm7 disrupts vesicle-associated resistance against the apoplastic pathogen L. maculans.

Ectopic Expression of the Executor-Type R Gene Paralog Xa27B in Rice Leads to Spontaneous Lesions and Enhanced Disease Resistance.

Plant disease resistance (R) gene-mediated effector-triggered immunity (ETI) is usually associated with hypersensitive response (HR) and provides robust and race-specific disease resistance against pathogenic infection. The activation of ETI and HR in plants is strictly regulated, and improper activation will lead to cell death. Xa27 is an executor-type R gene in rice induced by the TAL effector AvrXa27 and confers disease resistance to Xanthomonas oryzae pv. oryzae (Xoo). Here we reported the characterization of a transgenic line with lesion mimic phenotype, designated as Spotted leaf and resistance 1 (Slr1), which was derived from rice transformation with a genomic subclone located 5,125 bp downstream of the Xa27 gene. Slr1 develops spontaneous lesions on its leaves caused by cell death and confers disease resistance to both Xoo and Xanthomonas oryzae pv. oryzicola. Further investigation revealed that the Slr1 phenotype resulted from the ectopic expression of an Xa27 paralog gene, designated as Xa27B, in the inserted DNA fragment at the Slr1 locus driven by a truncated CaMV35Sx2 promoter in reverse orientation. Disease evaluation of IRBB27, IR24, and Xa27B mutants with Xoo strains expressing dTALE-Xa27B confirmed that Xa27B is a functional executor-type R gene. The functional XA27B-GFP protein was localized to the endoplasmic reticulum and apoplast. The identification of Xa27B as a new functional executor-type R gene provides additional genetic resources for studying the mechanism of executor-type R protein-mediated ETI and developing enhanced and broad-spectrum disease resistance to Xoo through promoter engineering. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Viral synergism suppresses R gene-mediated resistance by impairing downstream defense mechanisms in soybean.

Viral synergism occurs when mixed infection of a susceptible plant by 2 or more viruses leads to increased susceptibility to at least 1 of the viruses. However, the ability of 1 virus to suppress R gene-controlled resistance against another virus has never been reported. In soybean (Glycine max), extreme resistance (ER) against soybean mosaic virus (SMV), governed by the Rsv3 R-protein, manifests a swift asymptomatic resistance against the avirulent strain SMV-G5H. Still, the mechanism by which Rsv3 confers ER is not fully understood. Here, we show that viral synergism broke this resistance by impairing downstream defense mechanisms triggered by Rsv3 activation. We found that activation of the antiviral RNA-silencing pathway and the proimmune mitogen-activated protein kinase 3 (MAPK3), along with the suppression of the proviral MAPK6, are hallmarks of Rsv3-mediated ER against SMV-G5H. Surprisingly, infection with bean pod mottle virus (BPMV) disrupted this ER, allowing SMV-G5H to accumulate in Rsv3-containing plants. BPMV subverted downstream defenses by impairing the RNA-silencing pathway and activating MAPK6. Further, BPMV reduced the accumulation of virus-related siRNAs and increased the virus-activated siRNA that targeted several defense-related nucleotide-binding leucine-rich repeat receptor (NLR) genes through the action of the suppression of RNA-silencing activities encoded in its large and small coat protein subunits. These results illustrate that viral synergism can result from abolishing highly specific R gene resistance by impairing active mechanisms downstream of the R gene.

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Identification of Pseudo-R genes in Vitis vinifera and characterization of their role as immunomodulators in host-pathogen interactions.

INTRODUCTION: Duplication events are fundamental to co-evolution in host-pathogen interactions. Pseudogenes (Ψs) are dysfunctional paralogs of functional genes and resistance genes (Rs) in plants are the key to disarming pathogenic invasions. Thus, deciphering the roles of pseudo-R genes in plant defense is momentous. OBJECTIVES: This study aimed to functionally characterize diverse roles of the resistance Ψs as novel gene footprints and as significant gene regulators in the grapevine genome. METHODS: PlantPseudo pipeline and HMM-profiling identified whole-genome duplication-derived (WGD) Ψs associated with resistance genes (Ψ-Rs). Further, novel antifungal and antimicrobial peptides were characterized for fungal associations using protein-protein docking with Erysiphe necator proteins. miRNA and tasiRNA target sites and transcription factor (TF) binding sites were predicted in Ψ-Rs. Finally, differential co-expression patterns in Ψ-Rs-lncRNAs-coding genes were identified using the UPGMA method. RESULTS: 2,746 Ψ-Rs were identified from 31,032 WGD Ψs in the genome of grapevine. 69-antimicrobial and 81-antifungal novel peptides were generated from Ψ-Rs. The putative genic potential was predicted for five novel antifungal peptides which were further characterized by docking against E. necator proteins. 395 out of 527 resistance loci-specific Ψ-Rs were acting as parental gene mimics. Further, to explore the diverse roles of Ψ-Rs in plant-defense, we identified 37,026 TF-binding sites, 208 miRNA, and 99 tasiRNA targeting sites on these Ψ-Rs. 194 Ψ-Rs were exhibiting tissue-specific expression patterns. The co-expression network analysis between Ψs-lncRNA-genes revealed six out of 79 pathogen-responsive Ψ-Rs as significant during pathogen invasion. CONCLUSIONS: Our study provides pathogen responsive Ψ-Rs integral for pathogen invasion, which will offer a useful resource for future experimental validations. In addition, our findings on novel peptide generations from Ψ-Rs offer valuable insights which can serve as a useful resource for predicting novel genes with the futuristic potential of being investigated for their bioactivities in the plant system.

Genetic Analysis of HIV-1 vpr Sequences from HIV-Infected Older Patients on Long-Term Antiretroviral Therapy.

BACKGROUND: Many HIV-infected individuals have achieved undetectable viral load and increased CD4 T cell counts due to the success of Antiretroviral Therapy (ART). However, HIV persists in resting T cells, monocytes/macrophages and other quiescent cells. Furthermore, the HIV- 1 vpr accessory gene may play an important role in the persistence of HIV in these infected patients. OBJECTIVES: Therefore, we characterized the HIV-1 vpr gene from PBMC DNA of 14 HIV-infected older patients on long-term ART with mostly undetectable viral load and increased CD4 T cell counts. METHODS: Peripheral Blood Mononuclear Cells (PBMC) were isolated from 14 HIV-infected individuals, followed by extraction of genomic DNA, amplification of HIV-1 vpr gene by polymerase chain reaction (PCR), cloning of vpr gene in TOPO vector and characterization of correct size recombinant inserts containing vpr genes. An average of 13 clones were sequenced from each patient, followed by sequence analysis by bioinformatic tools. RESULTS: Phylogenetic analysis of 182 vpr sequences demonstrated that the vpr sequences of each patient were well separated and discriminated from other patients' sequences and formed distinct clusters. The vpr sequences showed a low degree of viral heterogeneity, lower estimates of genetic diversity and about half of the patients' sequences were under positive selection pressure. While the majority of the vpr deduced amino acid sequences from most patients contained intact open reading frames, several sequences, mostly from two patients, had stop codons. Numerous patient-specific and common amino acid motifs were found in deduced vpr sequences. The functional domains required for vpr activity, including virion incorporation, nuclear import of pre-integration complex and cell cycle arrest, were generally conserved in most vpr sequences. Several of the known Cytotoxic T-lymphocytes (CTL) epitopes in vpr showed variation in our patients' sequences. CONCLUSION: In summary, a low degree of genetic variability, conservation of functional domains and variations in CTL epitopes were the features of vpr sequences from the 14 HIV-infected older patients with controlled viremia on long-term ART.

Evolution of resistance (R) gene specificity.

Plant resistance (R) genes are members of large gene families with significant within and between species variation. It has been hypothesised that a variety of processes have shaped R gene evolution and the evolution of R gene specificity. In this review, we illustrate the main mechanisms that generate R gene diversity and provide examples of how they can change R gene specificity. Next, we explain which evolutionary mechanisms are at play and how they determine the fate of new R gene alleles and R genes. Finally, we place this in a larger context by comparing the diversity and evolution of R gene specificity within and between species scales.

R gene triplication confers European fodder turnip with improved clubroot resistance.

Clubroot is one of the most important diseases for many important cruciferous vegetables and oilseed crops worldwide. Different clubroot resistance (CR) loci have been identified from only limited species in Brassica, making it difficult to compare and utilize these loci. European fodder turnip ECD04 is considered one of the most valuable resources for CR breeding. To explore the genetic and evolutionary basis of CR in ECD04, we sequenced the genome of ECD04 using de novo assembly and identified 978 candidate R genes. Subsequently, the 28 published CR loci were physically mapped to 15 loci in the ECD04 genome, including 62 candidate CR genes. Among them, two CR genes, CRA3.7.1 and CRA8.2.4, were functionally validated. Phylogenetic analysis revealed that CRA3.7.1 and CRA8.2.4 originated from a common ancestor before the whole-genome triplication (WGT) event. In clubroot susceptible Brassica species, CR-gene homologues were affected by transposable element (TE) insertion, resulting in the loss of CR function. It can be concluded that the current functional CR genes in Brassica rapa and non-functional CR genes in other Brassica species were derived from a common ancestral gene before WGT. Finally, a hypothesis for CR gene evolution is proposed for further discussion.

In vitro Delivery of HIV-1 Nef-Vpr DNA Construct Using the Human Antimicrobial Peptide LL-37.

BACKGROUND AND OBJECTIVES: DNA-based therapeutic vaccines have been proposed as a promising strategy for the treatment of established HIV infections. However, these vaccines are often associated with certain shortcomings, such as poor immunogenicity and low transfection efficiency. In this study, we investigated the ability of LL-37 to deliver a potential immunogenic fusion construct comprising HIV-1 nef and vpr genes into a mammalian cell line. METHODS: First, the pEGFP-N1 eukaryotic expression vector harboring the HIV-1 nef-vpr fusion was produced free of endotoxin on a large scale. Then, DNA/LL-37 complexes were prepared by coincubation of pEGFP-nef-vpr with LL-37 for 45 minutes at different nitrogen to phosphate (N/P) ratios. The formation of DNA/peptide complexes was investigated by gel retardation assay. Next, the stability and morphological characteristics of the nanoparticles were evaluated. The toxicity of LL-37 and the nanoparticles in HEK-293T cells were assessed by MTT assay. The transfection efficiency of the DNA/LL-37 complexes was studied by fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS: LL-37 formed stable complexes with pEGFP-nef-vpr (diameter of 150-200 nm) while providing good protection against nucleolytic and proteolytic degradation. The peptide significantly affected cell viability even at low concentrations. However, the LL-37/DNA complexes had no significant cytotoxic effect. Treatment of cells with pEGFP-N1/LL-37 and pEGFP-nef-vpr/LL-37 resulted in transfection of 36.32% ± 1.13 and 25.55% ± 2.07 of cells, respectively. CONCLUSION: Given these findings and the important immunomodulatory and antiviral activities of LL- 37, the use of this peptide can be further exploited in the development of novel gene delivery strategies and vaccine design.

Characterization of APX and APX-R gene family in Brassica juncea and B. rapa for tolerance against abiotic stresses.

KEY MESSAGE: APX and APX-R gene families were identified and characterized in two important oilseed species of Brassica. Gene expression under abiotic stress conditions, recombinant protein expression, and analysis further divulged their drought, heat, and salt-responsive behavior. Ascorbate peroxidases (APX) are heme-dependent enzymes that rid the cells of H2O2 and regulate diverse biological processes. In the present study, we performed APX gene family characterization in two Brassica sp. (B. juncea and B. rapa) as these are commercially important oilseed crops and affected severely by abiotic stresses. We identified 16 BjuAPX and 9 BraAPX genes and 2 APX-R genes each in B. juncea and B. rapa genomes, respectively. Phylogenetic analysis divided the APX genes into five distinct clades, which exhibited conservation in the gene structure, motif organization, and sub-cellular location within the clade. Structural analysis of APX and APX-R proteins revealed the amino acid substitutions in conserved domains of APX-R proteins. The expression profiling of BjuAPX and BraAPX genes showed that 3 BjuAPX, 7BraAPX, and 2 BraAPX-R genes were drought and heat responsive. Notably, BjuAAPX1a, BjuAPX1d, BjuAAPX6, BraAAPX1a, BraAAPX2, and BraAAPX3b showed high expression levels in RT-qPCR. Cis-regulatory elements in APX and APX-R gene promoters supported the differential behavior of these genes. Further, two stress-responsive genes BjuAPX1d and BraAAPX2 were cloned, characterized, and their roles were validated under heat, drought, salt, and cold stress in bacterial expression system. This study for the first time reports the presence of APX activity in dimeric and LMW form of purified BraAAPX2 protein. The study may help pave way for developing abiotic stress-tolerant Brassica crops.

Pepper Mottle Virus and Its Host Interactions: Current State of Knowledge.

Pepper mottle virus (PepMoV) is a destructive pathogen that infects various solanaceous plants, including pepper, bell pepper, potato, and tomato. In this review, we summarize what is known about the molecular characteristics of PepMoV and its interactions with host plants. Comparisons of symptom variations caused by PepMoV isolates in plant hosts indicates a possible relationship between symptom development and genetic variation. Researchers have investigated the PepMoV-plant pathosystem to identify effective and durable genes that confer resistance to the pathogen. As a result, several recessive pvr or dominant Pvr resistance genes that confer resistance to PepMoV in pepper have been characterized. On the other hand, the molecular mechanisms underlying the interaction between these resistance genes and PepMoV-encoded genes remain largely unknown. Our understanding of the molecular interactions between PepMoV and host plants should be increased by reverse genetic approaches and comprehensive transcriptomic analyses of both the virus and the host genes.

Gum acacia PEG iron oxide nanocomposite (GA-PEG-IONC) induced pharmacotherapeutic activity on the Las R gene expression of Pseudomonas aeruginosa and HOXB13 expression of prostate cancer (Pc 3) cell line. A green therapeutic approach of molecular mechanism inhibition.

Among the diverse nanomaterials, polymer-based nanocomposites are gained more attention due to their high efficacy, target biological activities, biodegradability and biocompatibility-gum acacia (GA) - a polymer obtained from acacia trees-is considering the multifunctional nanocomposite synthesis. Distinctive Physico-chemical and biocompatibility properties of gum acacia are utilised to prepare a highly stable, biologically active, eco-friendly Nanocomposite. In this current investigation, gum acacia - poly ethylene glycol grafted iron oxide nanocomposite (GA-PEG-IONC) was synthesised by in situ green science principles. The synthesised Nanocomposite was evaluated against the molecular mechanism of urinary tract pathogenic bacterial strains and prostate cancer cells (Pc 3). Nanocomposite prepared in this examination exhibited notable structural, functional stability with nanoarchitecture which was affirmed by Fourier transform infrared spectroscopy (FTIR), electron microscopic studies, atomic force microscopy (AFM), vibrating sample magnetometric analysis (VSM) and X-ray diffraction (XRD), Synthesised Nanocomposite brought about notable antibacterial activity against urinary tract pathogenic strains by recording potential inhibitory effect on the expression of Las R gene. Inhibition of Las R gene expression reduced notable effect on biofilm development. Anticancer activity against prostate cancer cells (Pc3) was investigated by measurement of HOXB13 gene expression level. Inhibition of HOXB13 gene expression by the IONC brought about structural, functional changes. HOXB13 gene expression inhibition reveals a remarkable cytotoxic effect by recording decreased cell viability. Morphometric analysis by phase-contrast and DAPI fluorescence staining demonstrates that the Nanocomposite prompted cell morphology anomalies or apoptotic changes. Nanocomposite treatment brought about a good sign of Apoptosis by recording enhanced caspase 3 and 9 activities, DNA fragmentation and elevated reactive oxygen species generation (ROS). Hemocompatibility studies were carried out to determine the biocompatibility of the Nanocomposite. Spectrophotometric estimation of plasma haemoglobin, microscopic examination of whole blood cells shows the Nanocomposite was not inciting any indication of toxicity. These findings infer that IONC synthesised in the present study is the promising contender for a broad scope of biomedical applications, especially as an antibacterial and anticancer agent.

Plant death caused by inefficient induction of antiviral R-gene-mediated resistance may function as a suicidal population resistance mechanism.

Land plant genomes carry tens to hundreds of Resistance (R) genes to combat pathogens. The induction of antiviral R-gene-mediated resistance often results in a hypersensitive response (HR), which is characterized by virus containment in the initially infected tissues and programmed cell death (PCD) of the infected cells. Alternatively, systemic HR (SHR) is sometimes observed in certain R gene-virus combinations, such that the virus systemically infects the plant and PCD induction follows the spread of infection, resulting in systemic plant death. SHR has been suggested to be the result of inefficient resistance induction; however, no quantitative comparison has been performed to support this hypothesis. In this study, we report that the average number of viral genomes that establish cell infection decreased by 28.7% and 12.7% upon HR induction by wild-type cucumber mosaic virus and SHR induction by a single-amino acid variant, respectively. These results suggest that a small decrease in the level of resistance induction can change an HR to an SHR. Although SHR appears to be a failure of resistance at the individual level, our simulations imply that suicidal individual death in SHR may function as an antiviral mechanism at the population level, by protecting neighboring uninfected kin plants.

Eighty years of gene-for-gene relationship and its applications in identification and utilization of R genes.

The gene-for-gene relationship of host-pathogen interaction explained by H. H. Flor in mid of the 20th century set a milestone in understanding the biochemical and genetic basis of plant diseases and several components involved in plant-pathogen interactions. It highlighted the importance of accomplishing differential sets and understanding the pathogen population structure, it further led to the identification and cloning of several resistance (R) genes in plants. These R genes have been deployed and altered for fighting against diseases in a large number of crops using various conventional approaches and biotechnological tools. Identification of R genes and their corresponding Avr genes in many cases played a significant role in understanding of R-Avr gene interactions. Rapid cloning of R genes and editing of susceptible R genes are the other avenues that have broadened the horizon of utilizing R genes in crop improvement programmes. Further, combining R genes with quantitative disease resistance genes has paved the way to develop durable resistance in cultivars. The recent advances in genetics, genomics, bioinformatics and other OMICS tools are now providing greater prospects for deeper understanding of host-pathogen interaction.

Draft genome sequence of the pulse crop blackgram [Vigna mungo (L.) Hepper] reveals potential R-genes.

Blackgram [Vigna mungo (L.) Hepper] (2n = 2x = 22), an important Asiatic legume crop, is a major source of dietary protein for the predominantly vegetarian population. Here we construct a draft genome sequence of blackgram, for the first time, by employing hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. The final de novo whole genome of blackgram is ~ 475 Mb (82% of the genome) and has maximum scaffold length of 6.3 Mb with scaffold N50 of 1.42 Mb. Genome analysis identified 42,115 genes with mean coding sequence length of 1131 bp. Around 80.6% of predicted genes were annotated. Nearly half of the assembled sequence is composed of repetitive elements with retrotransposons as major (47.3% of genome) transposable elements, whereas, DNA transposons made up only 2.29% of the genome. A total of 166,014 SSRs, including 65,180 compound SSRs, were identified and primer pairs for 34,816 SSRs were designed. Out of the 33,959 proteins, 1659 proteins showed presence of R-gene related domains. KIN class was found in majority of the proteins (905) followed by RLK (239) and RLP (188). The genome sequence of blackgram will facilitate identification of agronomically important genes and accelerate the genetic improvement of blackgram.

Xa7, a new executor R gene that confers durable and broad-spectrum resistance to bacterial blight disease in rice.

Bacterial blight (BB) is a globally devastating rice disease caused by Xanthomonas oryzae pv. oryzae (Xoo). The use of disease resistance (R) genes in rice breeding is an effective and economical strategy for the control of this disease. Nevertheless, a majority of R genes lack durable resistance for long-term use under global warming conditions. Here, we report the isolation of a novel executor R gene, Xa7, that confers extremely durable, broad-spectrum, and heat-tolerant resistance to Xoo. The expression of Xa7 was induced by incompatible Xoo strains that secreted the transcription activator-like effector (TALE) AvrXa7 or PthXo3, which recognized effector binding elements (EBEs) in the Xa7 promoter. Furthermore, Xa7 induction was faster and stronger under high temperatures. Overexpression of Xa7 or co-transformation of Xa7 with avrXa7 triggered a hypersensitive response in plants. Constitutive expression of Xa7 activated a defense response in the absence of Xoo but inhibited the growth of transgenic rice plants. In addition, analysis of over 3000 rice varieties showed that the Xa7 locus was found primarily in the indica and aus subgroups. A variation consisting of an 11-bp insertion and a base substitution (G to T) was found in EBEAvrXa7 in the tested varieties, resulting in a loss of Xa7 BB resistance. Through a decade of effort, we have identified an important BB resistance gene and characterized its distinctive interaction with Xoo strains; these findings will greatly facilitate research on the molecular mechanism of Xa7-mediated resistance and promote the use of this valuable gene in breeding.

The Xa7 resistance gene guards the rice susceptibility gene SWEET14 against exploitation by the bacterial blight pathogen.

Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.

The dissection of R genes and locus Pc5.1 in Phytophthora capsici infection provides a novel view of disease resistance in peppers.

BACKGROUND: Phytophthora capsici root rot (PRR) is a disastrous disease in peppers (Capsicum spp.) caused by soilborne oomycete with typical symptoms of necrosis and constriction at the basal stem and consequent plant wilting. Most studies on the QTL mapping of P. capsici resistance suggested a consensus broad-spectrum QTL on chromosome 5 named Pc.5.1 regardless of P. capsici isolates and resistant resources. In addition, all these reports proposed NBS-ARC domain genes as candidate genes controlling resistance. RESULTS: We screened out 10 PRR-resistant resources from 160 Capsicum germplasm and inspected the response of locus Pc.5.1 and NBS-ARC genes during P. capsici infection by comparing the root transcriptomes of resistant pepper 305R and susceptible pepper 372S. To dissect the structure of Pc.5.1, we anchored genetic markers onto pepper genomic sequence and made an extended Pc5.1 (Ext-Pc5.1) located at 8.35 Mb-38.13 Mb on chromosome 5 which covered all Pc5.1 reported in publications. A total of 571 NBS-ARC genes were mined from the genome of pepper CM334 and 34 genes were significantly affected by P. capsici infection in either 305R or 372S. Only 5 inducible NBS-ARC genes had LRR domains and none of them was positioned at Ext-Pc5.1. Ext-Pc5.1 did show strong response to P. capsici infection and there were a total of 44 differentially expressed genes (DEGs), but no candidate genes proposed by previous publications was included. Snakin-1 (SN1), a well-known antimicrobial peptide gene located at Pc5.1, was significantly decreased in 372S but not in 305R. Moreover, there was an impressive upregulation of sugar pathway genes in 305R, which was confirmed by metabolite analysis of roots. The biological processes of histone methylation, histone phosphorylation, DNA methylation, and nucleosome assembly were strongly activated in 305R but not in 372S, indicating an epigenetic-related defense mechanism. CONCLUSIONS: Those NBS-ARC genes that were suggested to contribute to Pc5.1 in previous publications did not show any significant response in P. capsici infection and there were no significant differences of these genes in transcription levels between 305R and 372S. Other pathogen defense-related genes like SN1 might account for Pc5.1. Our study also proposed the important role of sugar and epigenetic regulation in the defense against P. capsici.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60 °C for 30 min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100 fg of template DNA compared to 1 ng and 10 pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.