The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants.

Ectopic expression of the Arabidopsis RESISTANCE TO POWDERY MILDEW8.1 (RPW8.1) boosts pattern-triggered immunity leading to enhanced resistance to different pathogens in Arabidopsis and rice. However, the underlying regulatory mechanism remains largely elusive. Here, we report that XAP5 CIRCADIAN TIMEKEEPER (XCT, At2g21150) positively regulates RPW8.1-mediated cell death and disease resistance. Forward genetic screen identified the b3-17 mutant that exhibited less cell death and susceptibility to powdery mildew and bacterial pathogens. Map-based cloning identified a G-to-A point mutation at the 3' splice site of the 8th intron, which resulted in splice shift to 8-bp down-stream of the original splice site of XCT in b3-17, and introduced into a stop codon after two codons leading to a truncated XCT. XCT has previously been identified as a circadian clock gene required for small RNA biogenesis and acting down-stream of ETHYLENE-INSENSITIVE3 (EIN3) in the ethylene-signaling pathway. Here we further showed that mutation or down-regulation of XCT by artificial microRNA reduced RPW8.1-mediated immunity in R1Y4, a transgenic line expressing RPW8.1-YFP from the RPW8.1 native promoter. On the contrary, overexpression of XCT in R1Y4 background enhanced RPW8.1-mediated cell death, H2O2 production and resistance against powdery mildew. Consistently, the expression of RPW8.1 was down- and up-regulated in xct mutant and XCT overexpression lines, respectively. Taken together, these results indicate that XCT positively regulates RPW8.1-mediated cell death and disease resistance, and provide new insight into the regulatory mechanism of RPW8.1-mediated immunity.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term "sequence-specific DNA binding transcription factor activity." We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

MicroRNAs fine-tune plant growth and resistance against multiple biotic and abiotic stresses. The trade-off between biomass and resistance can penalize crop yield. In this study, we have shown that rice miR530 regulates blast disease resistance, yield, and growth period. While the overexpression of miR530 results in compromised blast disease resistance, reduced grain yield, and late maturity, blocking miR530 using a target mimic (MIM530) leads to enhanced resistance, increased grain yield, and early maturity. Further study revealed that the accumulation of miR530 was decreased in both leaves and panicles along with the increase of age. Such expression patterns were accordant with the enhanced resistance from seedlings to adult plants, and the grain development from panicle formation to fully-filled seeds. Divergence analysis of miR530 precursor with upstream 1,000-bp promoter sequence in 11 rice species revealed that miR530 was diverse in Oryza sativa japonica and O. sativa indica group, which was consistent with the different accumulation of miR530 in japonica accessions and indica accessions. Altogether, our results indicate that miR530 coordinates rice resistance, yield, and maturity, thus providing a potential regulatory module for breeding programs aiming to improve yield and disease resistance.

Soybean (Glycine max) is an important economic crop that provides abundant oil and high quality protein for human beings. As the process of reproductive growth directly determines the crop seed yield and quality, we initiated studies to identify genes that regulate soybean floral organ development. One R2R3-MYB transcription factor gene, designated as GmMYB181, was found to be enriched in flowers based on microarray analysis and was further functionally investigated in transgenic Arabidopsis. GmMYB181 protein contains two MYB domains, which localized to the nucleus and displayed transcriptional activation in yeast hybrid system. Real-time quantitative PCR (qRT-PCR) results suggested GmMYB181 exclusively expressed in flower tissue. In Arabidopsis, overexpression of GmMYB181 altered the morphology of floral organs, fruit size and plant architecture, including outward curly sepals, smaller siliques, increased lateral branches and reduced plant height, indicating that GmMYB181 is involved in the development of reproductive organs and plays an important role in controlling plant architecture. Further, microarray analysis revealed that overexpressing GmMYB181 in Arabidopsis affected the expression of 3450 genes in mature flowers, including those involved in floral organ, seed/fruit development, and responded to different hormone signals.

Rice blast caused by Magnaporthe oryzae (M. oryzae) is a major threat to global rice production. In recent years, small interference RNAs (siRNAs) and host-induced gene silencing (HIGS) has been shown to be new strategies for the development of transgenic plants to control fungal diseases and proved a useful tool to study gene function in pathogens. We here tested whether in vitro feeding artificial siRNAs (asiRNAs) could compromise M. oryzae virulence and in vivo HIGS technique could improve rice blast resistance. Our data revealed that silencing of M. oryzae MoAP1 by feeding asiRNAs targeting MoAP1 (i.e., asiR1245, asiR1362, and asiR1115) resulted in inhibited fungal growth, abnormal spores, and decreased pathogenicity. Among the asiRNAs, asiR1115 was the most inhibitory toward the rice blast fungus. Conversely, the asiRNAs targeting three other genes (i.e., MoSSADH, MoACT, and MoSOM1) had no effect on fungal growth. Transgenic rice plants expressing RNA hairpins targeting MoAP1 exhibited improved resistance to 11 tested M. oryzae strains. Confocal microscopy also revealed profoundly restricted appressoria and mycelia in rice blast-infected transgenic rice plants. Our results demonstrate that in vitro asiRNA and in vivo HIGS were useful protection approaches that may be valuable to enhance rice blast resistance.

Rubisco activase (RCA), a key photosynthetic protein, catalyses the activation of Rubisco and thus plays an important role in photosynthesis. Although the RCA gene has been characterized in a variety of species, the molecular mechanism regulating its transcription remains unclear. Our previous studies on RCA gene expression in soybean suggested that expression of this gene is regulated by trans-acting factors. In the present study, we verified activity of the GmRCAα promoter in both soybean and Arabidopsis and used a yeast one-hybrid (Y1H) system for screening a leaf cDNA expression library to identify transcription factors (TFs) interacting with the GmRCAα promoter. Four basic leucine zipper (bZIP) TFs, GmbZIP04g, GmbZIP07g, GmbZIP1, and GmbZIP71, were isolated, and GmbZIP04g and GmbZIP07g were confirmed as able to bind to a 21-nt G-box-containing sequence. Additionally, the expression patterns of GmbZIP04g, GmbZIp07g, and GmRCAα were analyzed in response to abiotic stresses and during a 24-h period. Our study will help to advance elucidation of the network regulating GmRCAα transcription.

Timely prediction of seed viability loss over long-term storage represents a challenge in management and conservation of ex situ plant genetic resources. However, little attention has been paid to study the process of seed deterioration and seed aging signals under storage. An attempt was made here to investigate morphological and molecular changes in the scutellum and aleurone sections of naturally or artificially aged wheat seeds using TUNEL assay and DAPI staining. Twelve wheat genotypes or samples exposed to natural ageing (NA) or accelerated ageing (AA) were assayed and these samples had germination rates ranging from 11 to 93%. The assayed samples showed substantial changes in scutellum, but not aleurone. The nuclei observed in the majority of the scutellum cells of the NA seed samples of lower germination rates were longer in size and less visible, while the scutellum cell morphology or arrangement remained unchanged. In contrast, longer AA treatments resulted in the loss of scutellum cell structure, collapse of cell layers, and disappearance of honey comb arrangements. These nuclei and structural changes were consistent with the DNA assessments of nuclear alternations for the selected wheat samples. Interestingly, the sample seed germination loss was found to be associated with the reductions in the scutellum nuclear content and with the increases in the scutellum nuclei length to width ratio. These findings are significant for understanding the process of wheat seed deterioration and are also useful for searching for sensitive seed aging signals for developing tools to monitor seed viability under storage.

MADS-domain proteins are important transcription factors involved in many aspects of plant reproductive development. In this study, a MADS-box gene, Glycine max AGAMOUS-LIKE1 (GmAGL1), was isolated from soybean flower. The transcript of GmAGL1 was expressed in flowers and pods of different stages in soybean and was highly expressed in carpels. GmAGL1 is a nucleus-localized transcription factor and can interact directly with SEP-like proteins in soybean flowers. Ectopic overexpression of GmAGL1 resulted in the absence of petals in Arabidopsis. Moreover, morphological changes in the valves were observed in 35S:GmAGL1 Arabidopsis fruits that dehisced before the seeds reached full maturity. GmAGL1 was found to be sufficient to activate the expression of Arabidopsis ALC, IND, STK, SEP1, and SEP3. Therefore, our data suggest that GmAGL1 may play important roles in both floral organ identity and fruit dehiscence.

Fitness cost is a common phenomenon in rice blast disease-resistance breeding. MiR396 is a highly conserved microRNA (miRNA) family targeting Growth Regulating Factor (OsGRF) genes. Mutation at the target site of miR396 in certain OsGRF gene or blocking miR396 expression leads to increased grain yield. Here we demonstrated that fitness cost can be trade-off in miR396-OsGRFs module via balancing growth and immunity against the blast fungus. The accumulation of miR396 isoforms was significantly increased in a susceptible accession, but fluctuated in a resistant accession upon infection of Magnaporthe oryzae. The transgenic lines over-expressing different miR396 isoforms were highly susceptible to M. oryzae. In contrast, overexpressing target mimicry of miR396 to block its function led to enhanced resistance to M. oryzae in addition to improved yield traits. Moreover, transgenic plants overexpressing OsGRF6, OsGRF7, OsGRF8, and OsGRF9 exhibited enhanced resistance to M. oryzae, but showed different alteration of growth. While overexpression of OsGRF7 led to defects in growth, overexpression of OsGRF6, OsGRF8, and OsGRF9 resulted in better or no significant change of yield traits. Collectively, our results indicate that miR396 negatively regulates rice blast disease- resistance via suppressing multiple OsGRFs, which in turn differentially control growth and yield. Therefore, miR396-OsGRFs could be a potential module to demolish fitness cost in rice blast disease-resistance breeding.

Ustilaginoidea virens is a biotrophic fungal pathogen specifically colonizing rice floral organ and causes false smut disease of rice. This disease has emerged as a serious problem that hinders the application of high-yield rice cultivars, by reducing grain yield and quality as well as introducing mycotoxins. However, the pathogenic mechanisms of U. virens are still enigmatic. Here we demonstrate that U. virens employs a secreted protein UvCBP1 to manipulate plant immunity. In planta expression of UvCBP1 led to compromised chitin-induced defense responses in Arabidopsis and rice, including burst of reactive oxygen species (ROS), callose deposition, and expression of defense-related genes. In vitro-purified UvCBP1 protein competes with rice chitin receptor OsCEBiP to bind to free chitin, thus impairing chitin-triggered rice immunity. Moreover, UvCBP1 could significantly promote infection of U. virens in rice flowers. Our results uncover a mechanism of a floral fungus suppressing plant immunity and pinpoint a universal role of chitin-battlefield during plant-fungi interactions.

Pod dehiscence (shattering) is the main cause of serious yield loss during the soybean mechanical harvesting process. A better understanding of the genetic architecture and molecular mechanisms of pod dehiscence is of great significance for soybean breeding. In this study, genome-wide association analysis (GWAS) with NJAU 355K SoySNP array was performed to detect single nucleotide polymorphisms (SNPs) associated with pod dehiscence in an association panel containing 211 accessions across five environments. A total of 163 SNPs were identified as significantly associated with pod dehiscence. Among these markers, 136 SNPs identified on chromosome 16 were located in the known QTL qPDH1. One, one, three, eleven, three, one, three, three and one SNPs were distributed on chromosome 1, 4, 6, 8, 9, 11, 17, 18, and 20, respectively. Favorable SNPs and six haplotypes were identified based on ten functional SNPs; among those Hap2 and Hap3 were considered as optimal haplotypes. In addition, based on GWAS results, the candidate gene Glyma09g06290 was identified. Quantitative real-time PCR (qRT-PCR) results and polymorphism analysis suggested that Glyma09g06290 might be involved in pod dehiscence. Furthermore, a derived cleaved amplified polymorphic sequences (dCAPS) marker for Glyma09g06290 was developed. Overall, the loci and genes identified in this study will be helpful in breeding soybean accessions resistant to pod dehiscence.

A previous complementary cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis examined responses to the powdery mildew pathogen Oidium neolycopersici (On) of the resistant cultivar Solanum habrochiates G1.1560, carrying the Ol-1 resistance gene, and susceptible cultivar S. lycopersicum Moneymaker (MM). Among other findings, a differentially expressed transcript-derived fragment (DE-TDF) (M14E72-213) was upregulated in near isogenic line (NIL)-Ol-1, but absent in MM. This DE-TDF showed high homology to a gene of unknown function, which we named ShORR-1 (Solanum habrochaites Oidium Resistance Required-1). However, MM homolog of ShORR-1 (named ShORR-1-M) was still found with 95.26% nucleic acid sequence similarity to ShORR-1 from G1.1560 (named ShORR-1-G); this was because the cut sites of restriction enzymes in the previous complementary cDNA-AFLP analysis was absent in ShORR-1-M and differs at 13 amino acids from ShORR-1-G. Transient expression in onion epidermal cells showed that ShORR-1 is a membrane-localized protein. Virus-induced gene silencing (VIGS) of ShORR-1-G in G1.1560 plants increased susceptibility to On. Furthermore, overexpressing of ShORR-1-G conferred MM with resistance to On, involving extensive hydrogen peroxide accumulation and formation of abnormal haustoria. Knockdown of ShORR-1-M in MM did not affect its susceptibility to On, while overexpressing of ShORR-1-M enhanced MM's susceptibility to On. We also found that changes in transcript levels of six well-known hormone signaling and defense-related genes are involved in ShORR-1-G-mediated resistance to On. The results indicate that ShORR-1-M and ShORR-1-G have antagonistic effects in tomato responses to On, and that ShORR-1 is essential for Ol-1-mediated resistance in tomato.