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Abstract: Both plant and animal genomes encode proteins with nucleotide binding domains fused to leucine-rich repeat domains that are involved in responses to pathogens. While these domain structures are probably an example of convergent evolution, there are a number of similarities in the core mechanisms by which these proteins are regulated. [Copyright &y& Elsevier]

To study the mechanisms of inducible disease resistance in conifers, changes in transcript accumulation in roots of Norway spruce (Picea abies (L.) Karst.) seedlings exposed to the root rot pathogen Ceratobasidium bicorne Erikss. and Ryv. (anamorph: Rhizoctonia sp.) were monitored by differential display (DD). Because C. bicorne attacks root tips, a desiccation treatment was added to exclude genes induced by pathogen-related desiccation stress. The DD analysis was defined by the use of 11 sets of primers, covering about 5% of the transcriptome. A comparison of gene expression in control, desiccation- and pathogen-stressed roots revealed 36 pathogen-induced gene transcripts. Based on database searches, these transcripts were assigned to four groups originating from spruce mRNA (25 transcripts), rRNA (five transcripts), fungal mRNA (two transcripts) and currently unknown cDNAs (four transcripts). Real-time PCR was applied to verify and quantify pathogen-induced changes in transcript accumulation. Of the 18 transcripts tested, nine were verified to be Norway spruce gene transcripts up-regulated from 1.3- to 66-fold in the infected roots. Four germin-like protein isoforms, a peroxidase and a glutathione S-transferase, all implicated in oxidative processes, including the oxidative burst, were predicted from sequence similarity searches. Seven class IV chitinase isoforms implicated in fungal cell wall degradation and a nucleotide binding site-leucine rich repeat (NBS-LRR) disease resistance protein homologue related to pathogen recognition were identified. Several transcript species, such as the NBS-LRR homologue and the germin-like protein homologues, have not previously been identified as pathogen-inducible genes in gymnosperms. [ABSTRACT FROM PUBLISHER]

The activated disease resistance ( ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC–NBS–LRR genes. The NBS region of most NBS–LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC–NBS–LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS–LRR linker, which was conserved within the different CC–NBS–LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC–NBS–LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species. [ABSTRACT FROM AUTHOR]

Degenerate primers designed based on known resistant genes (R-genes) and resistance gene analogs (RGAs) were used in combinations to elucidate RGAs from Sorghum bicolor, cultivar M 35-1. Most of the previously tried primer combinations resulted in amplicons of expected 500–600 bp sizes in sorghum along with few novel combinations. Restriction analysis of PCR amplicons of expected size revealed a group of fragments present in a single band indicating the heterogeneous nature of the amplicon. Many of these were cloned and some were considered for analysis. The nucleotide sequence of different cloned fragments was done and their predicted amino acid sequences compared to each other and to the amino acid sequences of known R-genes revealed significant sequence similarity. A cluster analysis based on neighbor-joining (N-J) method was carried out using sorghum RGAs (SRGAs) together with several analogous known R-genes resulting in two major groups; cluster-I comprising only SRGAs and cluster-II comprised of known R-gene sequences along with three SRGAs. Further analysis clearly indicated similarity of SRGAs in overall sense with already known ones from other crop plants. These sequences can be used as guidelines to detect, map and eventually isolate numerous R-genes in sorghum. [ABSTRACT FROM AUTHOR]

Plant resistance to many types of pathogens and pests can be achieved by the presence of disease resistance (R) genes. The nucleotide binding site-leucine rich repeat (NBS-LRR) class of R-genes is the most commonly isolated class of R-genes and makes up a super-family, which is often arranged in the genome as large multi-gene clusters. The NBS domain of these genes can be targeted by polymerase chain reaction (PCR) amplification using degenerate primers. Previous studies have used PCR derived NBS sequences to investigate both ancient R-gene evolution and recent evolution within specific plant families. However, comparative studies with the Asteraceae family have largely been ignored. In this study, we address recent evolution of NBS sequences within the Asteraceae and extend the comparison to the Arabidopsis thaliana genome. Using multiple sets of primers, NBS fragments were amplified from genomic DNA of three species from the family Asteraceae: Helianthus annuus (sunflower), Lactuca sativa (lettuce), and Cichorium intybus (chicory). Analysis suggests that Asteraceae species share distinct families of R-genes, composed of genes related to both coiled-coil (CC) and toll-interleukin-receptor homology (TIR) domain containing NBS-LRR R-genes. Between the most closely related species, (lettuce and chicory) a striking similarity of CC subfamily composition was identified, while sunflower showed less similarity in structure. These sequences were also compared to the A. thaliana genome. Asteraceae NBS gene subfamilies appear to be distinct from Arabidopsis gene clades. These data suggest that NBS families in the Asteraceae family are ancient, but also that gene duplication and gene loss events occur and change the composition of these gene subfamilies over time. [Copyright &y& Elsevier]

The majority of known plant resistance genes encode proteins with conserved nucleotide-binding sites and leucine-rich repeats (NBS-LRR). Degenerate primers based on conserved NBS-LRR motifs were used to amplify analogues of resistance genes from the dicot sugar beet. Along with a cDNA library screen, the PCR screen identified 27 genomic and 12 expressed NBS-LRR RGAs (nlRGAs) sugar beet clones. The clones were classified into three subfamilies based on nucleotide sequence identity. Sequence analyses suggested that point mutations, such as nucleotide substitutions and insertion/deletions, are probably the primary source of diversity of sugar beet nlRGAs. A phylogenetic analysis revealed an ancestral relationship among sugar beet nlRGAs and resistance genes from various angiosperm species. One group appeared to share the same common ancestor as Prf, Rx, RPP8, and Mi, whereas the second group originated from the ancestral gene from which 12C1, Xa1, and Cre3 arose. The predicted protein products of the nlRGAs isolated in this study are all members of the non-TIR-type resistance gene subfamily and share strong sequence and structural similarities with non-TIR-type resistance proteins. No representatives of the TIR-type RGAs were detected either by PCR amplification using TIR type-specific primers or by in silico screening of more than 16,000 sugar beet ESTs. These findings suggest that TIR type of RGAs is absent from the sugar beet genome. The possible evolutionary loss of TIR type RGAs in the sugar beet is discussed. [ABSTRACT FROM AUTHOR]

Molecular communication between plants and potential pathogens determines the ultimate outcome of their interaction. The directed delivery of microbial molecules into and around the host cell, and the subsequent perception of these by the invaded plant tissue (or lack thereof), determines the difference between disease and disease resistance. In theory, any foreign molecule produced by an invading pathogen could act as an elicitor of the broad physiological and transcriptional re-programming indicative of a plant defense response. The diversity of elicitors recognized by plants seems to support this hypothesis. Additionally, these elicitors are often virulence factors from the pathogen recognized by the host. This recognition, though genetically as simple as a ligand-receptor interaction, may require additional host proteins that are the nominal targets of virulence factor action. Transduction of recognition probably requires regulated protein degradation and results in massive changes in cellular home-ostasis, including a programmed cell death known as the hypersensitive response that indicates a successful, if perhaps over-zealous, disease resistance response. [ABSTRACT FROM AUTHOR]

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 × Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24(t) to Pi-32(t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes. [ABSTRACT FROM AUTHOR]

A set of NBS-containing sequences was isolated from genomic DNA of two grape species (Vitis amurensis and Vitis riparia) and characterised in a panel of Vitis genotypes carrying different levels of resistance against downy mildew and other diseases. A PCR-mediated approach made use of degenerate primers designed on conserved regions encoding known R-genes, and provided the source for cloning grape analogous sequences. Cloned sequences were digested with ten endonucleases and 29 out of 71 putative recombinant clones, which showed unique restriction patterns, were sequenced. Using a threshold value of 40% identity, at least 12 grape NBS-sequences had a high overall similarity with known R-genes, such as the Arabidopsis gene RPS5 and the tobacco gene N. The presence of internal conserved motifs provided evidence that sequences isolated from grape may belong to the NBS-LRR gene family. A cluster analysis based on the deduced amino acid sequence and carried out on grape NBS-sequences, together with several analogous domains of known R-genes, classified grape sequences into three major groups. A grape sequence of each group was used as a probe on Southern blots with digested genomic DNA from resistant and susceptible grapes. One of the NBS-containing probes showed a clear-cut separation between resistant species and susceptible varieties. This evidence makes the probe a candidate marker for disease resistance genes in Vitis germplasm. [ABSTRACT FROM AUTHOR]

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitrobinding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies. [ABSTRACT FROM AUTHOR]

Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx tacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions. [ABSTRACT FROM AUTHOR]

Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed. [ABSTRACT FROM AUTHOR]

Pea (Pisum sativum L.) sequences that are analogous to the conserved nucleotide binding site (NBS) domain found in a number of plant disease resistance genes (R-genes) were cloned. Using redundant oligonucleotide primers and the polymerase chain reaction (PCR), we amplified nine pea sequences and characterised their sequences. The pea R-gene analog (RGA)- deduced amino acid sequences demonstrated significant sequence similarity with known R-gene sequences lodged in public databases. The genomic locations of eight of the pea RGAs were determined by linkage mapping. The eight RGAs identified ten loci that mapped to six linkage groups. In addition, the genomic organization of the RGAs was inferred. Both single-copy and multicopy sequence families were present among the RGAs, and the multicopy families occurred most often as tightly linked clusters of related sequences. Intraspecific copy number variability was observed in three of the RGA sequence families, suggesting that these sequence families are evolving rapidly. The genomic locations of the pea RGAs were compared with the locations of known pea R-genes and sym genes involved in the pea-rhizobia symbiosis. Two pea RGAs mapped in the genomic region containing a pea R-gene, Fw, and four pea RGAs mapped in regions of the genome containing sym genes. [ABSTRACT FROM AUTHOR]

The soybean aphid poses a severe threat to soybean quality and yield by sucking phloem sap and transmitting plant viruses. An early-maturing and highly resistant soybean landrace, Fangzheng Moshidou, with markedly reduced aphid colonization has been identified by screening of aphid-resistant soybean accessions. In a population derived from the cross of Fangzheng Moshidou with the susceptible cultivar Beifeng 9, resistance was conferred by a single dominant gene. Three linked markers, Satt114, Satt334, and Sct_033, on chromosome 13 were identified by bulked-segregant analysis. Additional simple-sequence repeat and single-nucleotide polymorphism (SNP) markers were developed for gene mapping. The resistance of Fangzheng Moshidou was fine-mapped to the interval between the SNP markers YCSNP20 and YCSNP80, corresponding to 152.8 kb in the Williams 82 assembly 2 genome. This region was near the reported loci Rag2 and Rag5 but did not overlap the interval containing them. A unique haplotype is described for Fangzheng Moshidou that distinguishes it from soybean accessions PI 587972, PI 594879, and PI 567301B in the interval containing Rag2 and Rag5 . These results indicate that Fangzheng Moshidou harbors a novel gene at a tightly linked resistance locus, designated as RagFMD . Fourteen candidate genes were annotated in the fine-mapping region, including seven NBS-LRR genes, which are usually considered resistance genes in plant defense. Most of these candidate genes showed variations distinguishing the resistant and susceptible parents and some genes also showed differences in expression between the two parental lines and at several times after aphid infestation. Isolation of RagFMD would advance the study of molecular mechanisms of soybean aphid resistance and contribute to precise selection of resistant soybeans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Liu, Tang, Wang, Diao, Su, Sun, Shang, Guo and Qiu.)

Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus. [ABSTRACT FROM AUTHOR]

Main Conclusion: We advance Ulva's genetic tractability and highlight its value as a model organism by characterizing its APAF1_C/WD40 domain-encoding gene, which belongs to a family that bears homology to R genes. The multicellular chlorophyte alga Ulva mutabilis (Ulvophyceae, Ulvales) is native to coastal ecosystems worldwide and attracts both high socio-economic and scientific interest. To further understand the genetic mechanisms that guide its biology, we present a protocol, based on adapter ligation-mediated PCR, for retrieving flanking sequences in U. mutabilis vector-insertion mutants. In the created insertional library, we identified a null mutant with an insertion in an apoptotic protease activating factor 1 helical domain (APAF1_C)/WD40 repeat domain-encoding gene. Protein domain architecture analysis combined with phylogenetic analysis revealed that this gene is a member of a subfamily that arose early in the evolution of green plants (Viridiplantae) through the acquisition of a gene that also encoded N-terminal nucleotide-binding adaptor shared by APAF-1, certain R-gene products and CED-4 (NB-ARC) and winged helix-like (WH-like) DNA-binding domains. Although phenotypic analysis revealed no mutant phenotype, gene expression levels in control plants correlated to the presence of bacterial symbionts, which U. mutabilis requires for proper morphogenesis. In addition, our analysis led to the discovery of a putative Ulva nucleotide-binding site and leucine-rich repeat (NBS-LRR) Resistance protein (R-protein), and we discuss how the emergence of these R proteins in green plants may be linked to the evolution of the APAF1_C/WD40 protein subfamily., (© 2022. The Author(s).)

MicroRNAs (miRNAs) are a class of 21-24 nucleotides (nt) noncoding small RNAs ubiquitously distributed across the plant kingdom. miR482/2118, one of the conserved miRNA superfamilies originating from gymnosperms, has divergent main functions in core-angiosperms. It mainly regulates NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT (NBS-LRR) genes in eudicots, functioning as an essential component in plant disease resistance; in contrast, it predominantly targets numerous long noncoding RNAs (lncRNAs) in monocot grasses, which are vital for plant reproduction. Usually, miR482/2118 is 22-nt in length, which can trigger the production of phased small interfering RNAs (phasiRNAs) after directed cleavage. PhasiRNAs instigated from target genes of miR482/2118 enhance their roles in corresponding biological processes by cis-regulation on cognate genes and expands their function to other pathways via trans activity on different genes. This review summarizes the origin, biogenesis, conservation, and evolutionary characteristics of the miR482/2118 superfamily and delineates its diverse functions in disease resistance, plant development, stress responses, etc., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Plant resistance genes (R-genes) drive the immune responses of crops against specific pathotypes of disease-causing organisms. Over time, genetic diversity in R-genes and R-pseudogenes has arisen among different rice varieties. This bioinformatics study was carried out to (i) predict the full sets of candidate nucleotide-binding site leucine-rich repeat (NLR) R-genes present in six rice genomes; (ii) detect variation within candidate R-genes; (iii) identify potential selectable markers within and near to LRR genes among 75 diverse indica rice genomes. Four high quality indica genomes, plus the standard japonica and indica reference genomes, were analysed with widely available bioinformatic tools to identify candidate R-genes and R-pseudogenes. They were detected in clusters, consistent with previous studies. BLAST analysis of cloned protein sequences of 31 R-gene loci gave confidence in this approach for detection of cloned NLR R-genes. Approximately 10% of candidate R-genes were located within 1 kb of a microsatellite (SSR) marker. Sequence comparisons among indica rice genomes detected SNPs or InDels in 334 candidate rice R-genes. There were significantly more SNPs and InDels within the identified NLR R-gene candidates than in other types of gene. The genome-wide locations of candidate R-genes and their associated markers are presented here for the potential future development of improved disease-resistant varieties. Limitations of in silico approaches used for R-gene discovery are discussed. [ABSTRACT FROM AUTHOR]

Rice false smut (RFS), an emerging major fungal disease worldwide caused by Ustilaginoidea virens, affects rice grain quality and yield. RFS cause 2.8-49% global yield loss depending upon disease severity and cultivars. In India, the yield loss due to RFS ranged from 2 to 75%. Identification of the genes or quantitative trait loci (QTLs) governing disease resistance would be of utmost importance towards mitigating the economic losses incurred due to RFS. Here, we report mapping of RFS resistance QTLs from a resistant breeding line RYT2668. The mapping population was evaluated for RFS resistance under the field condition in three cropping seasons 2013, 2015, and 2016. A positive correlation among infected panicle/plant, total smut ball/panicle, and disease score was observed in the years 2013, 2015, and the mean data. A total of seven QTLs were mapped on rice chromosomes 2, 4, 5, 7, and 9 using 2326 single nucleotide polymorphism markers. Of these, two QTLs, qRFSr5.3 and qRFSr7.1a, were associated with the infected panicle per plant, one QTL qRFsr9.1 with total smut ball per panicle, and four QTLs qRFSr2.2, qRFSr4.3, qRFSr5.4, and qRFSr7.1b with disease score. Among them, a novel QTL qRFSr9.1 on chromosome 9 exhibits the largest phenotypic effect. The prediction of putative candidate genes within the qRFSr9.1 revealed four nucleotide-binding sites-leucine-rich repeat (NBS-LRR) domain-containing disease resistance proteins. In summary, our findings mark the hotspot region of rice chromosomes carrying genes/QTLs for resistance to the RFS disease., (© 2021. Institute of Plant Genetics, Polish Academy of Sciences, Poznan.)

Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis ( Xam ) seriously affects cassava yield. Genes encoding nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are among the most important disease resistance genes in plants that are specifically involved in the response to diverse pathogens. However, the in vivo roles of NBS-LRR remain unclear in cassava ( Manihot esculenta ). In this study, we isolated four MeLRR genes and assessed their expression under salicylic acid (SA) treatment and Xam inoculation. Four MeLRR genes positively regulate cassava disease general resistance against Xam via virus-induced gene silencing (VIGS) and transient overexpression. During cassava- Xam interaction, MeLRRs positively regulated endogenous SA and reactive oxygen species (ROS) accumulation and pathogenesis-related gene 1 ( PR1 ) transcripts. Additionally, we revealed that MeLRRs positively regulated disease resistance in Arabidopsis . These pathogenic microorganisms include Pseudomonas syringae pv. tomato , Alternaria brassicicola , and Botrytis cinerea . Our findings shed light on the molecular mechanism underlying the regulation of cassava resistance against Xam inoculation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Ye, Liu, Sun, Li, Wu, Zhou and Wan.)

Cucumber powdery mildew caused by Sphaerotheca fuliginea is a leaf disease that seriously affects cucumber's yield and quality. This study aimed to report two nucleotide-binding site-leucine-rich repeats (NBS-LRR) genes CsRSF1 and CsRSF2, which participated in regulating the resistance of cucumber to S. fuliginea. The subcellular localization showed that the CsRSF1 protein was localized in the nucleus, cytoplasm, and cell membrane, while the CsRSF2 protein was localized in the cell membrane and cytoplasm. In addition, the transcript levels of CsRSF1 and CsRSF2 were different between resistant and susceptible cultivars after treatment with exogenous substances, such as abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), ethephon (ETH), gibberellin (GA) and hydrogen peroxide (H2O2). The expression analysis showed that the transcript levels of CsRSF1 and CsRSF2 were correlated with plant defense response against S. fuliginea. Moreover, the silencing of CsRSF1 and CsRSF2 impaired host resistance to S. fuliginea, but CsRSF1 and CsRSF2 overexpression improved resistance to S. fuliginea in cucumber. These results showed that CsRSF1 and CsRSF2 genes positively contributed to the resistance of cucumber to S. fuliginea. At the same time, CsRSF1 and CsRSF2 genes could also regulate the expression of defense-related genes. The findings of this study might help enhance the resistance of cucumber to S. fuliginea. [ABSTRACT FROM AUTHOR]

Nucleotide-binding site and leucine-rich repeat receptors (NLRs) play pivotal roles in plant immunity. The regulation of NLR stability is essential to ensure effective immunity, whereas the exact mechanism is largely unclear. The Arabidopsis CC-NBS-LRR protein L5 (At1g12290) can induce cell death in Nicotiana benthamiana, but not in Arabidopsis thaliana. We screened the interactors of L5 by yeast two-hybrid, and found that the BOI can interact with the CC domain of L5. Transiently expressed BOI reduced the protein level of L5, and suppressed the auoactivity of L5 in N. benthamiana. BOI can interact and ubiquitinate L5 in vitro, and mediate the proteasomal degradation of L5 in N. benthamiana and Arabidopsis. The Lys425 in the NBS domain of L5 is the critical unbiquitin site for the degradation. In conclusion, our results reveal a mechanism for the control of the stability of L5 protein and for the suppressed of L5-triggered cell death by a RING-type E3 ligase through the ubiquitin proteasome system., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ubiquitination is a pivotal post-translational modification that regulates turnover of nucleotide-binding site and leucine-rich repeat receptors (NLRs). As a RING-type E3 ligase, BOI (Botrytis susceptible1 interactor) has been reported to interact with different proteins, and function in the nucleus. New studies have identified that BOI can interact and ubiquitinate L5 (AT1G12290), a CC-NBS-LRR protein in vitro, and mediate the proteasomal degradation of L5 in Nicotiana benthamiana and Arabidopsis thaliana. However, there still remains an unanswered question about where the degradation occurs at the subcellular level. In this study, the ubiquitination of L5 by BOI was determined in N. benthamiana. Meanwhile, we discovered that BOI exhibited nucleocytoplasmic localization and mediated the degradation of the plasma membrane localized L5 outside the nucleus. BOI and its homologs BRG1 and BRG3 function redundantly in negatively regulate the protein level of L5. Overall, this report reveals BOI and its homologs have multiple targets and function at different subcellular locations., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Late blight (caused by Phytophthora infestans ) poses a serious threat to tomato production but the number of late blight resistance genes isolated from tomato is limited, making resistance gene mining a high research priority. In this study, highly resistant CLN2037E and susceptible No. 5 tomato inbred lines were used to identify late blight resistance genes. Using transcriptome sequencing, we discovered 36 differentially expressed genes (DEGs), including 21 nucleotide binding site-leucine-rich repeat and 15 pathogenesis-related (PR) disease resistance genes. Cluster analysis and real-time quantitative PCR showed that these 36 genes possessed similar expression patterns in different inbred lines after inoculation with P. infestans . Moreover, two PR genes with unique responses were chosen to verify their functions when exposed to P. infestans : Solyc08g080660 and Solyc08g080670 , both of which were thaumatin-like protein genes and were clustered in the tomato genome. Functions of these two genes were identified by gene overexpression and gene editing technology. Overexpression and knockout of single Solyc08g080660 and Solyc08g080670 corresponded to an increase and decrease in resistance to late blight, respectively, and Solyc08g080660 led to a greater change in disease resistance compared with Solyc08g080670 . Cotransformation of dual genes resulted in a much greater effect than any single gene. This study provides novel candidate resistance genes for tomato breeding against late blight and insights into the interaction mechanisms between tomato and P. infestans .

Identification of full length genes along with upstream regulatory elements is important to understand its expression. Here, we report preparation of high titre genomic library and identification of a genomic clone containing Pi-k h gene with its complete upstream and downstream sequences from the rice blast resistant line Tetep. Structural analysis of protein revealed that Pi-k h has a central nucleotide binding site domain, leucine-rich repeats domain and a unique zinc-finger domain. Comparative analysis of Pi-k h protein sequence showed 64% and 45% similarity with the protein sequences of rice blast resistance genes Pi-b and Pi-ta, respectively. [ABSTRACT FROM AUTHOR]

Xiaoyan6 (XY6) is a wheat ( Triticum aestivum ) cultivar possessing nonrace-specific high-temperature seedling plant (HTSP) resistance against stripe rust, caused by Puccinia striiformis f. sp. tritici . Previously, we identified one particular gene, TaRPS2 , for its involvement in the HTSP resistance. To elucidate the role of TaRPS2 in the HTSP resistance, we cloned the full length of TaRPS2 from XY6. The transcriptional expression of TaRPS2 was rapidly upregulated (19.11-fold) under the normal-high-normal temperature treatment that induces the HTSP resistance. The expression level of TaRPS2 in leaves was higher than that in the stems and roots. Quantification of the endogenous hormones in wheat leaves after P. striiformis f. sp. tritici inoculation showed that 1-aminocyclopropane-1-carboxylic acid, salicylic acid (SA), and jasmonic acid were involved in the HTSP resistance. In addition, detection of hydrogen peroxide (H 2 O 2 ) accumulation indicated that reactive oxygen species burst was also associated with the HTSP resistance. Two hours after exogenous H 2 O 2 treatment or 0.5 h after SA treatment, the expression level of TaRPS2 was increased by 2.66 and 2.35 times, respectively. The subcellular localization of enhanced green fluorescent protein-TaRPS2 fusion protein was in the nuclei and plasma membranes. Virus-induced gene silencing of TaRPS2 reduced the level of HTSP resistance in XY6. Compared with the nonsilenced leaves, the TaRPS2 -silenced leaves had the reduction of necrotic cells but a greater number of uredinia. These results indicated that TaRPS2 positively regulates the HTSP resistance of XY6 against P. striiformis f. sp. tritici and is related to the SA and H 2 O 2 signaling pathways.

Key Message: Novel disease resistance gene paralogues are generated by targeted chromosome cleavage of tandem duplicated NBS-LRR gene complexes and subsequent DNA repair in soybean. This study demonstrates accelerated diversification of innate immunity of plants using CRISPR. Nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) gene families are key components of effector-triggered immunity. They are often arranged in tandem duplicated arrays in the genome, a configuration that is conducive to recombinations that will lead to new, chimeric genes. These rearrangements have been recognized as major sources of novel disease resistance phenotypes. Targeted chromosome cleavage by CRISPR/Cas9 can conceivably induce rearrangements and thus emergence of new resistance gene paralogues. Two NBS-LRR families of soy have been selected to demonstrate this concept: a four-copy family in the Rpp1 region (Rpp1L) and a large, complex locus, Rps1 with 22 copies. Copy-number variations suggesting large-scale, CRISPR/Cas9-mediated chromosome rearrangements in the Rpp1L and Rps1 complexes were detected in up to 58.8% of progenies of primary transformants using droplet-digital PCR. Sequencing confirmed development of novel, chimeric paralogs with intact open reading frames. These novel paralogs may confer new disease resistance specificities. This method to diversify innate immunity of plants by genome editing is readily applicable to other disease resistance genes or other repetitive loci.