Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

CmERF1 acts as a positive regulator of fruits and leaves growth in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important horticultural and economic crop. ETHYLENE RESPONSE FACTOR1 (ERF1) plays an important role in regulating plant development, and the resistance to multiple biotic and abiotic stresses. In this study, developmental biology, molecular biology and biochemical assays were performed to explore the biological function of CmERF1 in melon. Abundant transcripts of CmERF1 were found in ovary at green-yellow bud (GYB) and rapid enlargement (ORE) stages. In CmERF1 promoter, the cis-regulatory elements for indoleacetic acid (IAA), methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA), light and low temperature responses were found. CmERF1 could be significantly induced by ethylene, IAA, MeJA, SA, ABA, and respond to continuous light and low temperature stresses in melon. Ectopic expression of CmERF1 increased the length of siliqua and carpopodium, and expanded the size of leaves in Arabidopsis. Knockdown of CmERF1 led to smaller ovary at anthesis, mature fruit and leaves in melon. In CmERF1-RNAi #2 plants, 75 genes were differently expressed compared with control, and the promoter regions of 28 differential expression genes (DEGs) contained the GCC-box (AGCCGCC) or DRE (A/GCCGAC) cis-acting elements of CmERF1. A homolog of cell division cycle protein 48 (CmCDC48) was proved to be the direct target of CmERF1 by the yeast one-hybrid assay and dual-luciferase (LUC) reporter (DLR) system. These results indicated that CmERF1 was able to promote the growth of fruits and leaves, and involved in multiple hormones and environmental signaling pathways in melon.

The GRAS protein OsDLA involves in brassinosteroid signalling and positively regulates blast resistance by forming a module with GSK2 and OsWRKY53 in rice.

Brassinosteroids (BRs) play a crucial role in shaping the architecture of rice (Oryza sativa) plants. However, the regulatory mechanism of BR signalling in rice immunity remains largely unexplored. Here we identify a rice mutant dla, which exhibits decreased leaf angles and is insensitive to 24-epiBL (a highly active synthetic BR), resembling the BR-deficient phenotype. The dla mutation caused by a T-DNA insertion in the OsDLA gene leads to downregulation of the causative gene. The OsDLA knockout plants display reduced leaf angles and less sensitivity to 24-epiBL. In addition, both dla mutant and OsDLA knockout plants are more susceptible to rice blast compared to the wild type. OsDLA is a GRAS transcription factor and interacts with the BR signalling core negative regulator, GSK2. GSK2 phosphorylates OsDLA for degradation via the 26S proteasome. The GSK2 RNAi line exhibits enhanced rice blast resistance, while the overexpression lines thereof show susceptibility to rice blast. Furthermore, we show that OsDLA interacts with and stabilizes the WRKY transcription factor OsWRKY53, which has been demonstrated to positively regulate BR signalling and blast resistance. OsWRKY53 directly binds the promoter of PBZ1 and activates its expression, and this activation can be enhanced by OsDLA. Together, our findings unravel a novel mechanism whereby the GSK2-OsDLA-OsWRKY53 module coordinates blast resistance and plant architecture via BR signalling in rice.

The Molecular Genetics and Genomics of Plant-Pathogen Interactions.

Plants have evolved an intricate immune system to protect themselves from potential pathogens [...].

Magnaporthe oryzae endoplasmic reticulum membrane complex regulates the biogenesis of membrane proteins for pathogenicity.

In eukaryotes, the majority of newly synthesized integral membrane proteins are inserted into the endoplasmic reticulum (ER) membrane before transferred to their functional sites. The conserved ER membrane complex (EMC) takes part in the insertion process for tail-anchored membrane proteins. However, the function of EMC in phytopathogenic fungi has not been characterized. Here, we report the identification and functional characterization of two EMC subunits MoEmc5 and MoEmc2 in Magnaporthe oryzae. The knockout mutants ΔMoemc5 and ΔMoemc2 exhibit substantial defect in autophagy, pathogenicity, cell wall integrity, and magnesium ion sensitivity. We demonstrate that the autophagy process was severely impaired in the ΔMoemc5 and ΔMoemc2 mutants because of the low-protein steady-state level of Atg9, the sole membrane-associated autophagy protein. Furthermore, the protein level of membrane proteins Chs4, Fks1, and MoMnr2 is also significantly reduced in the ΔMoemc5 and ΔMoemc2 strains, leading to their supersensitivity to Calcofluor white, Congo red, and magnesium. In addition, MoEmc5, but not MoEmc2, acts as a magnesium transporter independent of its EMC function. Magnaporthe oryzae EMC regulates the biogenesis of membrane proteins for autophagy and virulence; therefore, EMC subunits could be potential targets for fungicide design in the future.

Two Species of Stemphylium, S. astragali and S. henanense sp. nov., Causing Leaf and Stem Spot Disease on Astragalus sinicus in China.

Astragalus sinicus is a versatile legume crop, primarily utilized as a green manure in China. During 2020 and 2021, A. sinicus plants exhibiting dark brown or reddish-brown lesions or spots on leaves and stems were collected from fields in the Henan, Sichuan, and Guangxi provinces of China. Sixteen single-spore isolates were isolated from the infected leaf and stem tissue samples. Phylogenetic analyses based on the concatenated internal transcribed spacer, gapdh, and cmdA sequences indicated that 14 of them belong to Stemphylium astragali, whereas two isolates can be well separated from other known species in this genus. Based on the morphological characteristics and nucleotide polymorphisms with sister taxa, the two isolates were identified as a new species named S. henanense. Furthermore, pathogenicity assays showed that the S. astragali and S. henanense isolates caused leaf and stem spot symptoms on A. sinicus. Altogether, we describe a new species of Stemphylium (i.e., S. henanense sp. nov.) causing leaf spot disease of A. sinicus. In addition, this is the first report of S. astragali causing stem spot disease of A. sinicus.

The Hidden Truths of Fungal Virulence and Adaptation on Hosts: Unraveling the Conditional Dispensability of Minichromosomes in the Hemibiotrophic Colletotrichum Pathogens.

Colletotrichum spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core chromosomes and two to three minichromosomes. While the core chromosomes regulate fungal growth, development and virulence, the extent to which the minichromosomes are involved in these processes is still uncertain. Here, we discuss the minichromosomes of three hemibiotrophic Colletotrichum pathogens, i.e., C. graminicola, C. higginsianum and C. lentis. These minichromosomes are typically less than one megabase in length, characterized by containing higher repetitive DNA elements, lower GC content, higher frequency of repeat-induced point mutations (RIPMs) and sparse gene distribution. Molecular genetics and functional analyses have revealed that these pathogens harbor one conditionally dispensable minichromosome, which is dispensable for fungal growth and development but indispensable for fungal virulence on hosts. They appear to be strain-specific innovations and are highly compartmentalized into AT-rich and GC-rich blocks, resulting from RIPMs, which may help protect the conditionally dispensable minichromosomes from erosion of already scarce genes, thereby helping the Colletotrichum pathogens maintain adaptability on hosts. Overall, understanding the mechanisms underlying the conditional dispensability of these minichromosomes could lead to new strategies for controlling anthracnose disease in crops.

CsTFL1 inhibits determinate growth and terminal flower formation through interaction with CsNOT2a in cucumber.

Cucumber (Cucumis sativus L.) is an important vegetable crop that carries on vegetative growth and reproductive growth simultaneously. Indeterminate growth is favourable for fresh market under protected environments, whereas determinate growth is preferred for pickling cucumber in the once-over mechanical harvest system. The genetic basis of determinacy is largely unknown in cucumber. In this study, map-based cloning of the de locus showed that the determinate growth habit is caused by a non-synonymous SNP in CsTFL1CsTFL1 is expressed in the subapical regions of the shoot apical meristem, lateral meristem and young stems. Ectopic expression of CsTFL1 rescued the terminal flower phenotype in the Arabidopsis tfl1-11 mutant and delayed flowering in wild-type Arabidopsis Knockdown of CsTFL1 resulted in determinate growth and formation of terminal flowers in cucumber. Biochemical analyses indicated that CsTFL1 interacts with a homolog of the miRNA biogenesis gene CsNOT2a; CsNOT2a interacts with FDP. Cucumber CsFT directly interacts with CsNOT2a and CsFD, and CsFD interacts with two 14-3-3 proteins. These data suggest that CsTFL1 competes with CsFT for interaction with CsNOT2a-CsFDP to inhibit determinate growth and terminal flower formation in cucumber.

A Functional Allele of CsFUL1 Regulates Fruit Length through Repressing CsSUP and Inhibiting Auxin Transport in Cucumber.

Fruit length is a prominent agricultural trait during cucumber (Cucumis sativus) domestication and diversifying selection; however, the regulatory mechanisms of fruit elongation remain elusive. We identified two alleles of the FRUITFULL (FUL)-like MADS-box gene CsFUL1 with 3393C/A Single Nucleotide Polymorphism variation among 150 cucumber lines. Whereas CsFUL1A was specifically enriched in the long-fruited East Asian type cucumbers (China and Japan), the CsFUL1C allele was randomly distributed in cucumber populations, including wild and semiwild cucumbers. CsFUL1A knockdown led to further fruit elongation in cucumber, whereas elevated expression of CsFUL1A resulted in significantly shorter fruits. No effect on fruit elongation was detected when CsFUL1C expression was modulated, suggesting that CsFUL1A is a gain-of-function allele in long-fruited cucumber that acts as a repressor during diversifying selection of East Asian cucumbers. Furthermore, CsFUL1A binds to the CArG-box in the promoter region of SUPERMAN, a regulator of cell division and expansion, to repress its expression. Additionally, CsFUL1A inhibits the expression of auxin transporters PIN-FORMED1 (PIN1) and PIN7, resulting in decreases in auxin accumulation in fruits. Together, our work identifies an agriculturally important allele and suggests a strategy for manipulating fruit length in cucumber breeding that involves modulation of CsFUL1A expression.

First Report of Leaf Spot Disease Caused by Boeremia linicola on Trifolium repens in China.

Trifolium repens L. (White clover) a multipurpose legume crop, primarily utilized as a green manure in China. In March 2018, field investigations showed that a leaf spot disease occurred on T. repens in three fields with 50% to 80% incidence (50 plants in each field were investigated) in Nanchong City, Sichuan Province of China. Infected leaves showed symptoms of irregular dark brown spots in the center of leaves or at the leaf margins. Symptomatic leaves were surface sterilized with 3% NaClO for 3 min followed by 75% ethanol for 30 s and then rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar and incubated at 25°C in the dark. Four pure cultures (Y3-1; Y3-2; Y3-3; Y3-4) were obtained by single spore isolation. On oatmeal agar medium, a colony reached 57 mm diameter after 9 d in alternating light and dark at 25℃. Fimbriate aerial hypha were flat and compact with pale brown to dark brown color. Conidiogenous cells were hyaline, smooth, ampulliform to doliiform (n = 30), ranging from 4.1 to 10.5 µm long (6.6 ± 1.8 µm) × 2.2 to 5.7 µm wide (4.1 ± 0.8 µm). Conidia were ellipsoidal to cylindrical, hyaline, thin walled, smooth, aseptate, with 2 to 4 polar guttules (n = 50), ranging from 3.2 to 11.3 µm long (6.3 ± 1.1 µm) × 2.1 to 4.1 µm wide (2.8 ± 0.4 µm). Conidial matrix was whitish. Morphologically these isolates resembled species in Boeremia (Chen et al. 2015). Genomic DNA of each culture was extracted from mycelia using the quick and safe method (Chi et al. 2009). The 28S large subunit of nuclear ribosomal RNA (LSU) region, internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α (TEF 1-α), ß-Tubulin (TUB2) were amplified with corresponding primers (Carbone and Kohn 1999; Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester 1990; White et al. 1990; Woudenberg et al.2009). Sequences were deposited in GenBank with accession numbers: ON705759 to ON705766, ON734043 to ON734046 and ON841585 to ON841592. Phylogenetic analysis was conducted with combined sequences of the five loci using the maximum likelihood (ML) and the maximum parsimony (MP) methods. The four isolates and the extype strain of B. linicola (CBS 116.76) clustered together with high bootstrap support (BS) values (MLBS = 100; MPBS = 98). All sequences showed 100% identity to those of CBS 116.76, except the ITS region of our isolates (ON705759 to ON705762), which show 99.6% identity to that of CBS 116.76. Based on morphological characteristics and phylogenetic results, our isolates were identified as B. linicola, although the morphological characteristics of CBS 116.76 had not been characterized. To assess pathogenicity, a conidial suspension (approximately 105 CFU/mL) of isolate Y3-1 was sprayed on 1-month-old healthy plants in a greenhouse at 22℃ to 28℃. Plants sprayed with sterilized water were used as negative controls. The test was conducted three times, each with 3 plants. After 10 days, the leaves of the plants showed irregular brown lesions that were similar to the symptoms observed in the field, control plants remained healthy. The pathogen was reisolated and confirmed to be B. linicola, thus completing the verification of Koch's Postulates. Compared to B. exigua, a causal pathogen associated with leaf spot on white clover reported by Wang et al (Wang et al. 2021), B. linicola produced larger conidia, and the two species did not cluster together in the phylogenetic tree. To our knowledge, this is the first report of leaf spot disease caused by B. linicola on Trifolium repens in China.

First Report of Pyricularia oryzae Causing Blast on Palm Grass in China.

Palm grass (Setaria palmifolia) has been used as an ornamental plant and vegetable crop (Wu, 2009; Plarre, 1995). In June 2019, 2-10 mm severe leaf lesions with gray centers and brown-yellow edges were observed on the leaves of palm grass in Liuyang city (28°43'N, 114°12'E), Hunan province, China (Fig. 1A). Disease incidence on leaves was 20 - 40%. The infected leaves were collected and disinfected with 75% alcohol for 30 sec and 1% sodium hypochlorite for 1 min, followed by three rinses in sterilized ddH2O, dried on sterilized filter paper, and incubated on water agar for 48 h under continuous fluorescent light at 26℃. Then, typical pyriform and 2-septate conidia (23.97 - 30.37 × 7.42 - 9.98 µm, N = 30) appeared at the lesions (Fig. 1B). Four single-spore were captured, and then grew on oatmeal tomato agar for seven days under continuous fluorescent light at 26℃ to obtain four isolates (LY-ZY-7a, -7b, -9b and -9c) and produce conidia for inoculation tests. The colony morphology of LY-ZY-7b on OTA was gray and floccose, and the growth rate was 6.15 - 6.31 mm/d at 26 °C (Fig. 1C). Spores of LY-ZY-7b were washed off with sterilized ddH2O plus 0.025% Tween-20 to make spore suspensions. For scratch inoculation, 10 µL spore suspension (1 × 105 spores/mL) was inoculated on the wound scratched with a sterilized pin along the vein (3 mm × 3 mm) on palm grass middle leaf of 4-week-old seedlings. The inoculated leaves were sealed in a 15-cm Petri dish. For spray inoculation, 20 mL spore suspension (5 × 104 spores/mL) was made and sprayed on ten healthy palm grasses of 4-week-old seedlings. Plants used as negative controls were sprayed with sterilized ddH2O plus 0.025% Tween-20 (Liu et al. 2022; Zhang et al. 2014). After inoculation, all plants were put into transparent boxes to maintain > 95% humidity and covered with black plastic bags for one day. Then, the boxes containing the plants were placed in a growth chamber at 26°C (12 h light / 12 h darkness photoperiod). After six days, typical blast-type lesions with brown-yellow edges were visible on the leaves. Control plants did not show symptoms (Fig. 1D, 1E). Microscopical examination showed that the conidia and conidiophore recovered from the lesion of the inoculated plants have the same morphology as those recovered from natural infected tissues (Fig. 1F, 1G). The colony morphology of the pathogen isolated from the artificially inoculated tissue was consistent with that of isolate LY-ZY-7b (Fig. 1C). The spore suspension (5 × 104 spores/mL) of isolate LY-ZY-7b and one rice-infecting strain P131 (Yang et al., 2010) was made and sprayed onto 4-week-old seedlings of three rice cultivars. But unfortunately, isolate LY-ZY-7b could not cause any disease lesions on the tested rice cultivars, whereas strain P131 produced many typical blast lesions on rice leaves (Fig. 1H). Then, the fungal genetic identity of four isolates (LY-ZY-7a, -7b, -9b, and -9c) was confirmed by comparison of the sequence obtained from partial DNA of Actin (ACT), ITS, and RPB1 loci from our isolates and those previously published by Klaubauf et al. 2014. The nucleotide sequences of ACT, ITS, and RPB1 were submitted to GenBank ON228695-ON228697 (ACT), ON210978-ON210980 (ITS), ON228698-ON228701 (RPB1). A phylogenetic tree deduced from a maximum likelihood analysis based on combined ACT-ITS-RPB1 sequence data of Pyricularia showed that these four isolates (LY-ZY-7a, -7b, -9b, and -9c) clustered together on Pyricularia oryzae, with a high bootstrap support value (Fig. 2). Based on morphological characteristics and molecular phylogeny, these four isolates were identified as P. oryzae (Klaubauf et al. 2014; Qi et al. 2019). To our knowledge, this is the first report of blast disease on palm grass caused by P. oryzae in China, which will help develop disease management strategies against palm grass blast. Moreover, as a host of P. oryzae, palm grass might contribute as an inoculum source for blast diseases on cereal crops (such as rice, wheat, and barley) caused by P. oryzae in the field.

First Report of Didymella glomerata Causing Didymella Leaf Blight on Maize in China.

Maize (Zea mays L.) is a staple food crop worldwide. In July 2021, gray leaf blight was observed on maize leaves in a field located in Panjin (41°7'11.98" N, 122°4'14.57" E), Liaoning Province, China. Nearly 5% of the maize plants were affected in the field. The leaves of the affected plants showed oval to oblong, gray, sunken lesions with yellow or tan margins. The lesions were scattered all over the leaf surface; however, they were absent on the stalks and other parts of the affected plants. To isolate the pathogen, leaf discs (1.25 mm2) excised from the blight lesions were surface-sterilized with 70% ethanol for 30 seconds, followed by 20% NaOCl for 2 minutes and finally rinsed three times with sterilized water. The discs were cultured on potato dextrose agar (PDA) plates supplemented with streptomycin (100 mg/L) and incubated at 25oC under a 12-h photoperiod for 7 days. Six single spore isolates (two per sampled infected leaf) were purified from the PDA culture plates. The fungal colonies of three selected isolates (one per sampled infected leaf; Pj-1, Pj-2, and Pj-3) were dark brown on the PDA plates and devoid of aerial hyphae; all three isolates grew 11 mm/day on the PDA plates. The number of conidia produced by the isolates on the 6-cm PDA plates 7 days after incubation was ranged from 160 x 108 to 208 x 108 (n = 36). Conidia were hyaline, single-celled and ellipsoidal (3.35-3.56 µm [width] x 6.47-6.70 [length] µm; n = 36). To identify the pathogen, four loci, i.e., 28S subunit (large subunit [LSU]) of the nuclear ribosomal (nr) DNA, internal transcribed spacer (ITS) region (ITS1, 5.8S subunit of nrDNA, and ITS2), the second-largest subunit of RNA polymerase II (rpb2) and ß-tubulin (tub2) were amplified using the primer sets described in the study by Chen el al. 2015. BLASTn search against GenBank revealed that the four amplicon sequences originating from Pj-1, Pj-2, and Pj-3 showed 99-100% homology to the type strain CBS 528.66 of D. glomerata. A phylogenetic tree deduced from a maximum likelihood analysis of a concatenated MUSCLE-based alignment of LSU, ITS region, rpb2, and tub2 sequences of 12 isolates/strains showed that the Pj isolates clustered together with CBS 528.66, along with other D. glomerata isolates/strains, with a high bootstrap support value (i.e., 99). Based on both morphological characteristics and molecular phylogeny, Pj-1, Pj-2, and Pj-3 were identified as the D. glomerata isolates. Since the amplicon sequences of the three isolates were identical, only Pj-2 sequences were deposited in GenBank with accession numbers OM372474 (LSU), OK485138 (ITS), OM406188 (rpb2), and OK485135 (tub2). To confirm pathogenicity, 14-day-old plants (V3 growth stage) of a maize cultivar P178 were spray-inoculated with the Pj-2 conidia (1 x 107 conidia/mL) in a growth chamber. The inoculated leaves exhibited typical gray leaf blight lesions (similar to those detected in the maize field) 7 days post-inoculation at 25oC and 95-100% humidity under a 12-h photoperiod, whereas the leaves spray-inoculated with sterilized water remained healthy. The pathogenicity assay was repeated three times; the pathogen was re-isolated from the inoculated leaves each time and confirmed by the morphological characteristics and the molecular phylogeny based on the four loci to be D. glomerata, fulfilling Koch's postulates. This first report of D. glomerata causing Didymella leaf blight on maize will help develop robust disease management strategies against this emerging fungal pathogen.

An Improved Split-Ring Resonator-Based Sensor for Microfluidic Applications.

This study proposes an ultrahigh-sensitivity split-ring resonator-based microwave sensor for retrieving the complex permittivity of liquid samples. An interdigital capacitor structure was used to expand the sensing area and the sensitivity. A defected ground structure and A parallel dual split-ring resonator were introduced to improve the quality factor. A polydimethylsiloxane microfluidic channel substrate was placed above the interdigital capacitor structure. The channel route coincided with the interdigital gap to fully utilize the strong electric field. Ethanol-water solutions with varying ethanol fractions were injected into the channel as the testing liquid. It was demonstrated that the variation in resonant frequency can be used to retrieve the dielectric properties of liquid samples. The proposed sensor used a small liquid volume of ~0.68 µL and provided values in good agreement with the reference data.

The Rice Malectin Regulates Plant Cell Death and Disease Resistance by Participating in Glycoprotein Quality Control.

In animals, malectin is well known to play an essential role in endoplasmic reticulum quality control (ERQC) by interacting with ribophorin I, one unit of the oligosaccharyltransferase (OST) complex. However, the functions of malectin in plants remain largely unknown. Here, we demonstrate the rice OsMLD1 is an ER- and Golgi-associated malectin protein and physically interacts with rice homolog of ribophorin I (OsRpn1), and its disruption leads to spontaneous lesion mimic lesions, enhanced disease resistance, and prolonged ER stress. In addition, there are many more N-glycosites and N-glycoproteins identified from the mld1 mutant than wildtype. Furthermore, OsSERK1 and OsSERK2, which have more N-glycosites in mld1, were demonstrated to interact with OsMLD1. OsMLD1 can suppress OsSERK1- or OsSERK2-induced cell death. Thus, OsMLD1 may play a similar role to its mammalian homologs in glycoprotein quality control, thereby regulating cell death and immunity of rice, which uncovers the function of malectin in plants.

Prp19-associated splicing factor Cwf15 regulates fungal virulence and development in the rice blast fungus.

The splicing factor Cwf15 is an essential component of the Prp19-associated component of the spliceosome and regulates intron splicing in several model species, including yeasts and human cells. However, the roles of Cwf15 remain unexplored in plant pathogenic fungi. Here, we report that MoCWF15 in the rice blast fungus Magnaporthe oryzae is non-essential to viability and important to fungal virulence, growth and conidiation. MoCwf15 contains a putative nuclear localization signal (NLS) and is localized into the nucleus. The NLS sequence but not the predicted phosphorylation site or two sumoylation sites was essential for the biological functions of MoCwf15. Importantly, MoCwf15 physically interacted with the Prp19-associated splicing factors MoCwf4, MoSsa1 and MoCyp1, and negatively regulated protein accumulations of MoCyp1 and MoCwf4. Furthermore, with the deletion of MoCWF15, aberrant intron splicing occurred in near 400 genes, 20 of which were important to the fungal development and virulence. Taken together, MoCWF15 regulates fungal growth and infection-related development by modulating the intron splicing efficiency of a subset of genes in the rice blast fungus.

OsNBL3, a mitochondrion-localized pentatricopeptide repeat protein, is involved in splicing nad5 intron 4 and its disruption causes lesion mimic phenotype with enhanced resistance to biotic and abiotic stresses.

Lesion mimic mutants are used to elucidate mechanisms controlling plant responses to pathogen attacks and environmental stresses. Although dozens of genes had been functionally demonstrated to be involved in lesion mimic phenotype in several plant species, the molecular mechanisms underlying the hypersensitive response are largely unknown. Here, a rice (Oryza sativa) lesion mimic mutant natural blight leaf 3 (nbl3) was identified from T-DNA insertion lines. The causative gene, OsNBL3, encodes a mitochondrion-localized pentatricopeptide repeat (PPR) protein. The nbl3 mutant exhibited spontaneous cell death response and H2 O2 accumulation, and displayed enhanced resistance to the fungal and bacterial pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. This resistance was consistent with the up-regulation of several defence-related genes; thus, defence responses were induced in nbl3. RNA interference lines of OsNBL3 exhibited enhanced disease resistance similar to that of nbl3, while the disease resistance in overexpression lines did not differ from that of the wild type. In addition, nbl3 displayed improved tolerance to salt, accompanied by up-regulation of several salt-associated marker genes. OsNBL3 was found to mainly participate in the splicing of mitochondrial gene nad5 intron 4. Disruption of OsNBL3 leads to the reduction in complex I activity, the elevation of alternative respiratory pathways and the destruction of mitochondrial morphology. Overall, the results demonstrated that the PPR protein-coding gene OsNBL3 is essential for mitochondrial development and functions, and its disruption causes the lesion mimic phenotype and enhances disease resistance and tolerance to salt in rice.

Methodological investigation into the noise influence on nanolasers' large signal modulation.

Nanolasers are considered ideal candidates for communications and data processing at the chip-level thanks to their extremely reduced footprint, low thermal load and potentially outstanding modulation bandwidth, which in some cases has been numerically estimated to exceed hundreds of GHz. The few experimental implementations reported to date, however, have so-far fallen very short of such predictions, whether because of technical difficulties or of overoptimistic numerical results. We propose a methodology to study the physical characteristics which determine the system's robustness and apply it to a general model, using numerical simulations of large-signal modulation. Changing the DC pump values and modulation frequencies, we further investigate the influence of intrinsic noise, considering, in addition, the role of cavity losses. Our results confirm that significant modulation bandwidths can be achieved, at the expense of large pump values, while the often targeted low bias operation is strongly noise- and bandwidth-limited. This fundamental investigation suggests that technological efforts should be oriented towards enabling large pump rates in nanolasers, whose performance promises to surpass microdevices in the same range of photon flux and input energy.

A novel glycine-rich domain protein, GRDP1, functions as a critical feedback regulator for controlling cell death and disease resistance in rice.

Lesion mimic mutants constitute a valuable genetic resource for unraveling the signaling pathways and molecular mechanisms governing the programmed cell death and defense responses of plants. Here, we identified a lesion mimic mutant, spl-D, from T-DNA insertion rice lines. The mutant exhibited higher accumulation of H2O2, spontaneous cell death, decreased chlorophyll content, up-regulation of defense-related genes, and enhanced disease resistance. The causative gene, OsGRDP1, encodes a cytosol- and membrane-associated glycine-rich domain protein. OsGRDP1 was expressed constitutively in all of the organs of the wild-type plant, but was up-regulated throughout plant development in the spl-D mutant. Both the overexpression and knockdown (RNAi) of OsGRDP1 resulted in the lesion mimic phenotype. Moreover, the intact-protein level of OsGRDP1 was reduced in the spotted leaves from both overexpression and RNAi plants, suggesting that the disruption of intact OsGRDP1 is responsible for lesion formation. OsGRDP1 interacted with an aspartic proteinase, OsAP25. In the spl-D and overexpression plants, proteinase activity was elevated, and lesion formation was partially suppressed by an aspartic proteinase inhibitor. Taken together, our results reveal that OsGRDP1 is a critical feedback regulator, thus contributing to the elucidation of the mechanism underlying cell death and disease resistance.

Structural basis of dimerization and dual W-box DNA recognition by rice WRKY domain.

In rice, the critical regulator of the salicylic acid signalling pathway is OsWRKY45, a transcription factor (TF) of the WRKY TF family that functions by binding to the W-box of gene promoters, but the structural basis of OsWRKY45/W-box DNA recognition is unknown. Here, we show the crystal structure of the DNA binding domain of OsWRKY45 (OsWRKY45-DBD, i.e. the WRKY and zinc finger domain) in complex with a W-box DNA. Surprisingly, two OsWRKY45-DBD molecules exchange ß4-ß5 strands to form a dimer. The domain swapping occurs at the hinge region between the ß3 and ß4 strands, and is bridged and stabilized by zinc ion via coordinating residues from different chains. The dimer contains two identical DNA binding domains that interact with the major groove of W-box DNA. In addition to hydrophobic and direct hydrogen bonds, water mediated hydrogen bonds are also involved in base-specific interaction between protein and DNA. Finally, we discussed the cause and consequence of domain swapping of OsWRKY45-DBD, and based on our work and that of previous studies present a detailed mechanism of W-box recognition by WRKY TFs. This work reveals a novel dimerization and DNA-binding mode of WRKY TFs, and an intricate picture of the WRKY/W-box DNA recognition.

LtEPG1, a Secretory Endopolygalacturonase Protein, Regulates the Virulence of Lasiodiplodia theobromae in Vitis vinifera and Is Recognized as a Microbe-Associated Molecular Patterns.

The Lasiodiplodia theobromae genome encodes numerous glycoside hydrolases involved in organic matter degradation and conducive to pathogen infection, whereas their molecular mechanisms are still largely unknown. Here, we identified the glycoside hydrolase family 28 endopolygalacturonase LtEPG1 in L. theobromae and characterized its function in detail. LtEPG1 acts as a virulence factor during L. theobromae infection. Overexpression and silencing of LtEPG1 in L. theobromae led to significantly increased and decreased lesion areas, respectively. Further, the high transcript level of LtEPG1 during the infection process supported its virulence function. Polygalacturonase activity of LtEPG1 was substantiated by detecting its ability to degrade pectin. Furthermore, LtEPG1 functioned as microbe-associated molecular patterns during the infection process. Both transient expression of LtEPG1 in planta and infiltration of purified LtEPG1 triggered cell death in Nicotiana benthamiana. Site-directed mutation of LtEPG1 indicated that the enzymatic activity of LtEPG1 is independent from its elicitor activity. A protein kinase, KINß1, was shown to interact in the yeast two-hybrid system with LtEPG1. This interaction was further confirmed in vitro using a pull-down assay. Our data indicate that LtEPG1 functions as a polygalacturonase and also serves as an elicitor with two independent mechanisms. Moreover, LtEPG1 may be able to manipulate host immune responses by regulating the KINß1-mediated signal pathway and consequently promote its own successful infection and symptom development.