The OsBDR1-MPK3 module negatively regulates blast resistance by suppressing the jasmonate signaling and terpenoid biosynthesis pathway.

Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae (M. oryzae) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae-induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae-elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic (allene oxide cyclase (AOC)/signaling (MYC2) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae, and their biosynthesis genes (TPS3 and TPS29) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.

Effect of Different Durations of Eye-Covering Pretreatment on Emergence Delirium after Ophthalmic Surgery in Preschool-Aged Children: A Randomized Controlled Study.

Background: Preoperative eye-covering training for 3 hours has been reported to effectively reduce the incidence of emergence delirium (ED) in preschool children. However, most children can only maintain the eye being covered for less than 60 min, and shortening eye-covering duration can also achieve similar clinical effects as long duration of eye-covering. This study was designed to compare the effects of 30-min and 60-min eye-covering pretreatment based on cartoon education only on preoperative anxiety, postoperative ED, and pain score after ophthalmic surgery with general anesthesia in preschool-aged children. Methods: Preschool-aged children (3-7 years) who were diagnosed with cataract, blepharoptosis, trichiasis, strabismus, eyelid tumor, and underwent ophthalmic surgery with general anesthesia from August 2021 to January 2022 were recruited. A total of 228 patients were randomly assigned at a 1 : 1:1 ratio to receive 30-min eye covering (30-min group), 60-min eye covering (60-min group) pretreatment, or programmed education only (C group). The preoperative anxiety, postoperative emergence delirium, and pain were compared between the groups. Results: The preoperative anxiety score, postoperative ED score, and incidence of ED in the 30-min group (n = 76) and 60-min group (n = 72) were significantly lower than those in the C group (n = 76), demonstrating a significant between-group difference (P < 0.001). However, the 30-min group and 60-min group had no significant difference in the abovementioned outcome measures (P > 0.05). Moreover, no significant difference was found in postoperative pain scores among the three groups (H = 0.274, P=0.872). Conclusion: Both 30-min and 60-min eye-covering pretreatments significantly reduce preoperative anxiety and postoperative ED after ophthalmic surgery with general anesthesia in preschool-aged children. The effects of the two groups show no intergroup difference, but the 30-min eye-covering pretreatment may be more convenient for practicing. Trial Registration. This study was registered with the No. NCT04973150.

Characterization of Agrobacterium-mediated co-transformation events in rice using green and red fluorescent proteins.

BACKGROUND: Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency. METHODS AND RESULTS: We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures. CONCLUSION: Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme.

Increasing Taxa Sampling Provides New Insights on the Phylogenetic Relationship Between Eriobotrya and Rhaphiolepis.

Eriobotrya (Rosaceae) is an economically important genus with around 30 species. It is widely distributed in tropical and warm temperate regions of Asia, with most of its species in China, Myanmar, and Vietnam. However, Eriobotrya is often confused with the smaller genus Rhaphiolepis, and the phylogenetic relationships between the two genera are controversial. Here we present phylogenetic analyses of 38 newly generated Eriobotrya and Rhaphiolepis nrDNA together with 16 sequences of nrDNA and 28 sequences of ITS obtained from GenBank, representing 28 species of Eriobotrya and 12 species of Rhaphiolepis, in order to reconstruct highly supported relationships for the two genera. Contrary to previous research based on limited sampling, our results highlight the monophyly of Eriobotrya as well as Rhaphiolepis. The topology recovered here is consistent with key morphological synapomorphies such as the persistent sepals in Eriobotrya. Our findings show that increased sampling of taxa can provide a more robust phylogeny through reducing phylogenetic error and increasing overall phylogenetic accuracy.

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System.

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

The complete plastome sequence of a subtropical tree Pyrus betulaefolia (Rosaceae).

The genus Pyrus, comprising several popular fruit crops worldwide, includes over 30 tree species. Here we determined the complete plastid genome sequence of Pyrus betulaefolia. The plastome consists of 160,184 bp, including a pair of inverted repeats (IRs) with a length of 26,384 bp separated by a large single-copy region (LSC) and a small single-copy region (SSC) of 88,121 bp and 19,295 bp, respectively. Further phylogenetic analyze was conducted using 11 complete plastid genomes of Rosaceae with KVM + F + I model, which supports Pyrus betulaefolia as a sister to all other eight Pyrus taxa with published plastomes.

Long Non-coding RNAs Responsive to Blast Fungus Infection in Rice.

BACKGROUND: Long non-coding RNAs (LncRNAs) have emerged as important regulators in many physiological processes in plant. By high-throughput RNA-sequencing, many pathogen-associated LncRNAs were mapped in various plants, and some of them were proved to be involved in plant defense responses. The rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most destructive diseases in rice. However, M. oryzae-induced LncRNAs in rice is yet to be studied. FINDINGS: We investigated rice LncRNAs that were associated with the rice blast fungus. Totally 83 LncRNAs were up-regulated after blast fungus infection and 78 were down-regulated. Of them, the natural antisense transcripts (NATs) were the most abundant. The expression of some LncRNAs has similar pattern with their host genes or neighboring genes, suggesting a cis function of them in regulating gene transcription level. The deferentially expressed (DE) LncRNAs and genes co-expression analysis revealed some LncRNAs were associated with genes known to be involved in pathogen resistance, and these genes were enriched in terpenoid biosynthesis and defense response by Gene Ontology (GO) enrichment analysis. Interestingly, one of up-regulated DE-intronic RNA was derived from a jasmonate (JA) biosynthetic gene, lipoxygenase RLL (LOX-RLL). Levels of JAs were significantly increased after blast fungus infection. Given that JA is known to regulate blast resistance in rice, we suggested that LncRNA may be involved in JA-mediated rice resistance to blast fungus. CONCLUSIONS: This study identified blast fungus-responsive LncRNAs in rice, which provides another layer of candidates that regulate rice and blast fungus interactions.

Complete plastome of a subtropical tree Eriobotrya malipoensis (Rosaceae) in Yunnan.

Eriobotrya malipoensis Kuan is an important wild woody evergreen tree within the genus Eriobotrya Lindl belonging the family Rosaceae. To better determine its phylogenetic location with respect to the other Eriobotrya species, the complete plastome of E. malipoensis was sequenced. The whole plastome is 159,313 bp in length, consisting of a pair of inverted repeat (IR) regions of 26,344 bp, one large single-copy (LSC) region of 87,270 bp, and one small single-copy (SSC) region of 19,355 bp. The overall G + C content of the whole plastome is 36.7%. Further, maximum likelihood phylogenetic analyse (TVM + F+R2 model) was conducted using 14 complete plastome of the Rosaceae. Our phylogeny supports the relationships: sisterhood of the E. malipoensis and E. fragrans Champ, flowed E. japonica Lindl.

The complete chloroplast genome sequence of Eriobotrya fragrans.

Eriobotrya fragrans Champion ex Bentham is a potential medicinal plant of the genus Eriobotrya Lindl in the family Rosaceae. To better determine its phylogenetic location with respect to the other Rosaceae species, the complete chloroplast genome of E. fragrans was sequenced. The whole E. fragrans chloroplast genome is 159,286 bp in length, consisting of a pair of inverted repeat (IR) regions of 26,343 bp, one large single-copy (LSC) region of 87,301 bp, and one small single-copy (SSC) region of 19,299 bp. The overall GC content of the whole chloroplast genome is 36.7%. Further, phylogenetic analysis using maximum likelihood with TVM + F+R2 model strongly supports the relationship: sisterhood of E. fragrans and E. japonica, followed by three species of Pyrus L.

Transcriptome Analysis Highlights Defense and Signaling Pathways Mediated by Rice pi21 Gene with Partial Resistance to Magnaporthe oryzae.

Rice blast disease is one of the most destructive rice diseases worldwide. The pi21 gene confers partial and durable resistance to Magnaporthe oryzae. However, little is known regarding the molecular mechanisms of resistance mediated by the loss-of-function of Pi21. In this study, comparative transcriptome profiling of the Pi21-RNAi transgenic rice line and Nipponbare with M. oryzae infection at different time points (0, 12, 24, 48, and 72 hpi) were investigated using RNA sequencing. The results generated 43,222 unique genes mapped to the rice genome. In total, 1109 differentially expressed genes (DEGs) were identified between the Pi21-RNAi line and Nipponbare with M. oryzae infection, with 103, 281, 209, 69, and 678 DEGs at 0, 12, 24, 48, and 72 hpi, respectively. Functional analysis showed that most of the DEGs were involved in metabolism, transport, signaling, and defense. Among the genes assigned to plant-pathogen interaction, we identified 43 receptor kinase genes associated with pathogen-associated molecular pattern recognition and calcium ion influx. The expression levels of brassinolide-insensitive 1, flagellin sensitive 2, and elongation factor Tu receptor, ethylene (ET) biosynthesis and signaling genes, were higher in the Pi21-RNAi line than Nipponbare. This suggested that there was a more robust PTI response in Pi21-RNAi plants and that ET signaling was important to rice blast resistance. We also identified 53 transcription factor genes, including WRKY, NAC, DOF, and ERF families that show differential expression between the two genotypes. This study highlights possible candidate genes that may serve a function in the partial rice blast resistance mediated by the loss-of-function of Pi21 and increase our understanding of the molecular mechanisms involved in partial resistance against M. oryzae.

Dissection of the genetic architecture of rice resistance to the blast fungus Magnaporthe oryzae.

Resistance in rice cultivars to the rice blast fungus Magnaporthe oryzae is complex and is controlled by both major genes and quantitative trait loci (QTLs). We undertook a genome-wide association study (GWAS) using the rice diversity panel 1 (RDP1) that was genotyped using a high-density (700 000 single nucleotide polymorphisms) array and inoculated with five diverse M. oryzae isolates. We identified 97 loci associated with blast resistance (LABRs). Among them, 82 were new regions and 15 co-localized with known blast resistance loci. The top 72 LABRs explained up to 98% of the phenotypic variation. The candidate genes in the LABRs encode nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance proteins, receptor-like protein kinases, transcription factors and defence-related proteins. Among them, LABR_64 was strongly associated with resistance to all five isolates. We analysed the function of candidate genes underlying LABR_64 using RNA interference (RNAi) technology and identified two new resistance alleles at the Pi5 locus. We demonstrate an efficient strategy for rapid allele discovery using the power of GWAS, coupled with RNAi technology, for the dissection of complex blast resistance in rice.

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct.

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a -46 minimal CaMV 35S promoter. The results of a transient expression assay showed that -46 minimal promoter::Cre recombinase (-46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and ß-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T(1) transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T(2) generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the -46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.

Construction and application of efficient Ac-Ds transposon tagging vectors in rice.

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element (HPT-Ds). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.

A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants.

Transposon insertional mutagenesis is an effective alternative to T-DNA mutagenesis when transformation through tissue culture is inefficient as is the case for many crop species. When used as activation tags, transposons can be exploited to generate novel gain-of-function phenotypes without transformation and are of particular value in the study of polyploid plants where gene knockouts will not have phenotypes. We have developed an in cis-activation-tagging Ac-Ds transposon system in which a T-DNA vector carries a Dissociation (Ds) element containing 4x cauliflower mosaic virus enhancers along with the Activator (Ac) transposase gene. Stable Ds insertions were selected using green fluorescent protein and red fluorescent protein genes driven by promoters that are functional in maize (Zea mays) and rice (Oryza sativa). The system has been tested in rice, where 638 stable Ds insertions were selected from an initial set of 26 primary transformants. By analysis of 311 flanking sequences mapped to the rice genome, we could demonstrate the wide distribution of the elements over the rice chromosomes. Enhanced expression of rice genes adjacent to Ds insertions was detected in the insertion lines using semiquantitative reverse transcription-PCR method. The in cis-two-element vector system requires minimal number of primary transformants and eliminates the need for crossing, while the use of fluorescent markers instead of antibiotic or herbicide resistance increases the applicability to other plants and eliminates problems with escapes. Because Ac-Ds has been shown to transpose widely in the plant kingdom, the activation vector system developed in this study should be of utility more generally to other monocots.

The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea.

The rice blast resistance (R) genes Pi2 and Piz-t confer broad-spectrum resistance against different sets of Magnaporthe grisea isolates. We first identified the Pi2 gene using a map-based cloning strategy. The Pi2 gene is a member of a gene cluster comprising nine gene members (named Nbs1-Pi2 to Nbs9-Pi2) and encodes a protein with a nucleotide-binding site and leucine-rich repeat (LRR) domain. Fine genetic mapping, molecular characterization of the Pi2 susceptible mutants, and complementation tests indicated that Nbs4-Pi2 is the Pi2 gene. The Piz-t gene, a Pi2 allele in the rice cultivar Toride 1, was isolated based on the Pi2 sequence information. Complementation tests confirmed that the family member Nbs4-Piz-t is Piz-t. Sequence comparison revealed that only eight amino-acid changes, which are confined within three consecutive LRR, differentiate Piz-t from Pi2. Of the eight variants, only one locates within the xxLxLxx motif. A reciprocal exchange of the single amino acid between Pi2 and Piz-t did not convert the resistance specificity to each other but, rather, abolished the function of both resistance proteins. These results indicate that the single amino acid in the xxLxLxx motif may be critical for maintaining the recognition surface of Pi2 and Piz-t to their respective avirulence proteins.

The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice.

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9-Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS-LRR gene cluster. Semiquantitative reverse transcriptase (RT)-PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity.

The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.