AGAMOUS-LIKE67 Cooperates with the Histone Mark Reader EBS to Modulate Seed Germination under High Temperature.

Seed germination is a vital developmental process that is tightly controlled by environmental signals, ensuring germination under favorable conditions. High temperature (HT) suppresses seed germination. This process, known as thermoinhibition, is achieved by activating abscisic acid and inhibiting gibberellic acid biosynthesis. The zinc-finger protein SOMNUS (SOM) participates in thermoinhibition of seed germination by altering gibberellic acid/abscisic acid metabolism, but the underlying regulatory mechanism is poorly understood. In this study, we report that SOM binds to its own promoter and activates its own expression in Arabidopsis (Arabidopsis thaliana) and identify the MADS-box transcription factor AGAMOUS-LIKE67 (AGL67) as a critical player in SOM function, based on its ability to recognize CArG-boxes within the SOM promoter and mediate the trans-activation of SOM under HTs. In addition, AGL67 recruits the histone mark reader EARLY BOLTING IN SHORT DAY (EBS), which recognizes H3K4me3 at SOM chromatin. In response to HTs, AGL67 and EBS are highly enriched around the SOM promoter. The AGL67-EBS complex is also necessary for histone H4K5 acetylation, which activates SOM expression, ultimately inhibiting seed germination. Taken together, our results reveal an essential mechanism in which AGL67 cooperates with the histone mark reader EBS, which bridges the process of H3K4me3 recognition with H4K5 acetylation, thereby epigenetically activating SOM expression to suppress seed germination under HT stress.

Loss function of SL (sekiguchi lesion) in the rice cultivar Minghui 86 leads to enhanced resistance to (hemi)biotrophic pathogens.

BACKGROUND: Serotonin, originally identified as a neurotransmitter in mammals, functions as an antioxidant to scavenge cellular ROS in plants. In rice, the conversion of tryptamine to serotonin is catalyzed by SL (sekiguchi lesion), a member of cytochrome P450 monooxygenase family. The sl mutant, originated from rice cultivar Sekiguchi-asahi, exhibits spontaneous lesions, whereas its immune responses to pathogens have not been clearly characterized. RESULTS: Here we identified three allelic mutants of SL in an indica rice restore line Minghui 86 (MH86), named as sl-MH-1, - 2 and - 3, all of which present the typical lesions under normal growth condition. Compared with those in MH86, the serotonin content in sl-MH-1 is dramatically decreased, whereas the levels of tryptamine and L-trytophan are significantly increased. The sl-MH-1 mutant accumulates high H2O2 level at its lesion sites and is more sensitive to exogenous H2O2 treatment than the wild type. When treated with the reductant vitamin C (Vc), the lesion formation on sl-MH-1 leaves could be efficiently suppressed. In addition, sl-MH-1 displayed more resistant to both the blast fungus and blight bacteria, Pyricularia oryzae (P. oryzae, teleomorph: Magnaporthe oryzae) and Xanthomonas oryzae pv. Oryzae (Xoo), respectively. The pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses, like reactive oxygen species (ROS) burst and callose deposition, were enhanced in sl-MH-1. Moreover, loss function of SL resulted in higher resting levels of the defense hormones, salicylic acid and jasmonic acid. The RNA-seq analysis indicated that after P. oryzae infection, transcription of the genes involved in reduction-oxidation regulation was the most markedly changed in sl-MH-1, compared with MH86. CONCLUSIONS: Our results indicate that SL, involving in the final step of serotonin biosynthesis, negatively regulates rice resistance against (hemi)biotrophic pathogens via compromising the PTI responses and defense hormones accumulation.

Exploring the Distribution of Blast Resistance Alleles at the Pi2/9 Locus in Major Rice-Producing Areas of China by a Novel Indel Marker.

Rice blast disease caused by the fungus Magnaporthe oryzae damages cereal crops and poses a high risk to rice production around the world. Currently, planting cultivars with resistance (R) genes is still the most environment-friendly approach to control this disease. Effective identification of R genes existing in diverse rice cultivars is important for understanding the distribution of R genes and predicting their contribution to resistance against blast isolates in regional breeding. Here, we developed a new insertion/deletion (InDel) marker, Pigm/2/9InDel, that can differentiate the cloned R genes (Pigm, Pi9, and Pi2/Piz-t) at the Pi2/9 locus. Pigm/2/9InDel combined with the marker Pi2-LRR for Pi2 was applied to determine the distribution of these four R genes among 905 rice varieties, most of which were collected from the major rice-producing regions in China. In brief, nine Pigm-containing varieties from Fujian and Guangdong provinces were identified. All of the 62 Pi2-containing varieties were collected from Guangdong, and 60 varieties containing Piz-t were from seven provinces. However, Pi9 was not found in any of the Chinese varieties. The newly identified varieties carrying the Pi2/9 alleles were further subjected to inoculation tests with regional blast isolates and field trials. Our results indicate that Pigm and Pi2 alleles have been introgressed for blast resistance breeding mainly in the Fujian and Guangdong region, and Pi9 is a valuable blast resistance resource to be introduced into China.

Bulked Segregant Analysis Coupled with Whole-Genome Sequencing (BSA-Seq) Mapping Identifies a Novel pi21 Haplotype Conferring Basal Resistance to Rice Blast Disease.

Basal or partial resistance has been considered race-non-specific and broad-spectrum. Therefore, the identification of genes or quantitative trait loci (QTLs) conferring basal resistance and germplasm containing them is of significance in breeding crops with durable resistance. In this study, we performed a bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) to identify QTLs controlling basal resistance to blast disease in an F2 population derived from two rice varieties, 02428 and LiXinGeng (LXG), which differ significantly in basal resistance to rice blast. Four candidate QTLs, qBBR-4, qBBR-7, qBBR-8, and qBBR-11, were mapped on chromosomes 4, 7, 8, and 11, respectively. Allelic and genotypic association analyses identified a novel haplotype of the durable blast resistance gene pi21 carrying double deletions of 30 bp and 33 bp in 02428 (pi21-2428) as a candidate gene of qBBR-4. We further assessed haplotypes of Pi21 in 325 rice accessions, and identified 11 haplotypes among the accessions, of which eight were novel types. While the resistant pi21 gene was found only in japonica before, three Chinese indica varieties, ShuHui881, Yong4, and ZhengDa4Hao, were detected carrying the resistant pi21-2428 allele. The pi21-2428 allele and pi21-2428-containing rice germplasm, thus, provide valuable resources for breeding rice varieties, especially indica rice varieties, with durable resistance to blast disease. Our results also lay the foundation for further identification and functional characterization of the other three QTLs to better understand the molecular mechanisms underlying rice basal resistance to blast disease.

The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii.

MAIN CONCLUSION: Nitric oxide signal and GSNOR activity play an essential role for Chlamydomonas reinhardtii response to salt stress. The unicellular alga Chlamydomonas reinhardtii is one of the most important model organisms phylogenetically situated between higher plants and animals. In the present study, we used comparative proteomics and physiological approaches to study the mechanisms underlying the response to salt stress in C. reinhardtii. We identified 74 proteins that accumulated differentially after salt stress, including oxidative enzymes and enzymes associated with nitric oxide (NO) metabolism, cell damage, and cell autophagy processes. A set of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR) activity, were induced to balance the cellular redox status during short-term salt stress. Enzymes involved in DNA repair and cell autophagy also contribute to adaptation to short-term salt stress. However, under long-term salt stress, antioxidant enzymes and GSNOR were gradually inactivated through protein S-nitrosylation, leading to oxidative damage and a reduction in cell viability. Modulating the protein S-nitrosylation levels by suppressing GSNOR activity or adding thioredoxin affected the plant's adaptation to salt stress, through altering the redox status and DNA damage and autophagy levels. Based on these data, we propose that unicellular algae use multiple strategies to adapt to salt stress, and that, during this process, GSNOR activity and protein S-nitrosylation levels play important roles.

Genome-Wide Identification and Analysis of Maize DnaJ Family Genes in Response to Salt, Heat, and Cold at the Seedling Stage.

DnaJ proteins, also known as HSP40s, play a key role in plant growth and development, and response to environmental stress. However, little comprehensive research has been conducted on the DnaJ gene family in maize. Here, we identify 91 ZmDnaJ genes from maize, which are likely distributed in the chloroplast, nucleus, and cytoplasm. Our analysis revealed that ZmDnaJs were classified into three types, with conserved protein motifs and gene structures within the same type, particularly among members of the same subfamily. Gene duplication events have likely contributed to the expansion of the ZmDnaJ family in maize. Analysis of cis-regulatory elements in ZmDnaJ promoters suggested involvement in stress responses, growth and development, and phytohormone sensitivity in maize. Specifically, four cis-acting regulatory elements associated with stress responses and phytohormone regulation indicated a role in adaptation. RNA-seq analysis showed constitutive expression of most ZmDnaJ genes, some specifically in pollen and endosperm. More importantly, certain genes also responded to salt, heat, and cold stresses, indicating potential interaction between stress regulatory networks. Furthermore, early responses to heat stress varied among five inbred lines, with upregulation of almost tested ZmDnaJ genes in B73 and B104 after 6 h, and fewer genes upregulated in QB1314, MD108, and Zheng58. After 72 h, most ZmDnaJ genes in the heat-sensitive inbred lines (B73 and B104) returned to normal levels, while many genes, including ZmDnaJ55, 79, 88, 90, and 91, remained upregulated in the heat-tolerant inbred lines (QB1314, MD108, and Zheng58) suggesting a synergistic function for prolonged protection against heat stress. In conclusion, our study provides a comprehensive analysis of the ZmDnaJ family in maize and demonstrates a correlation between heat stress tolerance and the regulation of gene expression within this family. These offer a theoretical basis for future functional validation of these genes.

Turnip mosaic virus NIb weakens the function of eukaryotic translation initiation factor 6 facilitating viral infection in Nicotiana benthamiana.

Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti-defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)-infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR-mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV-infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA-dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant-virus interaction.

Comparative proteome analyses reveal that nitric oxide is an important signal molecule in the response of rice to aluminum toxicity.

Acidic soils inhibit crop yield and reduce grain quality. One of the major contributing factors to acidic soil is the presence of soluble aluminum (Al(3+)) ions, but the mechanisms underlying plant responses to Al(3+) toxicity remain elusive. Nitric oxide (NO) is an important messenger and participates in various plant physiological responses. Here, we demonstrate that Al(3+) induced an increase of NO in rice seedlings; adding exogenous NO alleviated the Al(3+) toxicity related to rice growth and photosynthetic capacity, effects that could be reversed by suppressing NO metabolism. Comparative proteomic analyses successfully identified 92 proteins that showed differential expression after Al(3+) or NO treatment. In particular, some of the proteins are involved in reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism. Further analyses confirmed that NO treatment reduced Al(3+)-induced ROS and RNS toxicities by increasing the activities and protein expression of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR). Suppressing GSNOR enzymatic activity aggravated Al(3+) damage to rice and increased the accumulation of RNS. NO treatment altered the expression of proteins associated with cell wall synthesis, cell division and cell structure, calcium signaling and defense responses. On the basis of these results, we propose that NO activates multiple pathways that enhance rice adaptation to Al(3+) toxicity. Such findings may be applicable to crop engineering to enhance yield and improve stress tolerance.

iTRAQ-based protein profiling and functional identification of four genes involved in rice basal resistance against Magnaporthe oryzae in two contrasting rice genotypes.

Rice blast, caused by Magnaporthe oryzae, is one of the most destructive rice diseases. Developing blast-resistant rice cultivars represents the most economical and environmentally friend strategy for managing the disease. In our previous study, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative protein quantification was carried out to investigate the resistance gene Piz-t gene-mediated resistance response to infection in two contrasting rice genotypes of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and its wild-type Nipponbare (NPB). Here, from the comparisons of differentially expressed proteins (DEPs) of NPB-Piz-t to the avirulent isolate KJ201 (KJ201-Piz-t)and the virulent isolate RB22 (RB22-Piz-t) with mock-treated NPB-Piz-t (Mock-Piz-t), NPB to the virulent isolate KJ201(KJ201-NPB) and RB22 (RB22-NPB) with mock-treated NPB (Mock-NPB), 1, 1, and 6 common DEPs were, respectively, identified at 24, 48 and 72 h post-inoculation (hpi) in the susceptible comparisons of RB22-Pizt/Mock-Piz-t, KJ201-NPB/Mock-NPB, and RB22-NPB/Mock-NPB, involving in gi|54,290,836 and gi|59,800,021 were identified in the resistance comparison KJ201-Piz-t/Mock-Piz-t at 48 and 72 hpi respectively. Moreover, four genes of Os01g0138900 (gi|54,290,836), Os04g0659300 (gi|59,800,021), Os09g0315700 (gi|125,563,186) or Os04g0394200 (gi|21,740,743) were knocked out or overexpressed in NPB using gene over-expression and CRISPR/Cas9 technology, and results verified that the Os01g0138900 obviously affected the rice blast resistance. Further, expression and targeted metabolomics analysis illuminated the resistance response of cysteine-containing substances as gi|59,800,021 under blast infection. These results provide new targets for basal resistance gene identification and open avenues for developing novel rice blast resistant materials.

Association analysis of rice resistance genes and blast fungal avirulence genes for effective breeding resistance cultivars.

Utilization of rice blast-resistance (R) genes is the most economical and environmentally friendly method to control blast disease. However, rice varieties with R genes influence the outcome of genetic architectures of Magnaporthe oryzae (M. oryzae), and mutations in avirulence (AVR) genes of M. oryzae may cause dysfunction of the corresponding R genes in rice varieties. Although monitoring and characterizing rice R genes and pathogen AVR genes in field populations may facilitate the implementation of effective R genes, little is known about the changes of R genes over time and their ultimate impact on pathogen AVR genes. In this study, 117 main cultivated rice varieties over the past five decades and 35 M. oryzae isolates collected from those diseased plants were analyzed by PCR using gene-specific markers of the nine R genes and six primer pairs targeting the coding sequence or promoter of AVR genes, respectively. The R genes Pigm, Pi9, Pi2, Piz-t, Pi-ta, Pik, Pi1, Pikp, and Pikm were identified in 5, 0, 1, 4, 18, 0, 2, 1, and 0 cultivars, respectively. Significantly, none of these R genes had significant changes that correlated to their application periods of time. Among the four identified AVR genes, AVR-Pik had the highest amplification frequency (97.14%) followed by AVR-Pita (51.43%) and AVR-Pi9 (48.57%); AVR-Piz-t had the lowest frequency (28.57%). All these AVR genes except AVR-Pi9 had 1-2 variants. Inoculation mono-genic lines contained functional genes of Pi2/9 and Pik loci with 14 representative isolates from those 35 ones revealed that the presence of certain AVR-Piz-t, AVR-Pita variants, and AVR-Pik-E + AVR-Pik-D in M. oryzae populations, and these variants negated the ability of the corresponding R genes to confer resistance. Importantly, Pi2, Pi9, and Pigm conferred broad-spectrum resistance to these local isolates. These findings reveal that the complex genetic basis of M. oryzae and some effective blast R genes should be considered in future rice blast-resistance breeding programs.

Comparative Expression Profiling of Snf2 Family Genes During Reproductive Development and Stress Responses in Rice.

Sucrose non-fermenting 2 (Snf2) protein family, as chromatin remodeling factors, is an enormous and the most diverse protein family, which contributes to biological processes of replication, transcription, and DNA repair using the energy of adenosine triphosphate (ATP) hydrolysis. The members of Snf2 family proteins have been well characterized in Arabidopsis, rice, and tomato. Although this family received significant attention, few genes were identified uniquely for their roles in mediating reproductive development and stress tolerance in rice. In the present study, we comprehensively analyzed the expression profiling of Snf2 genes during reproductive development and biotic/abiotic stresses. Our results showed that five proteins (OsCHR712/715/720/726/739) were mainly localized in the nucleus, while OsCHR715/739 were also slightly expressed in the cell membrane. There were abundant cis-acting elements in the putative promoter of Snf2 genes, including dehydration, MeJA, MYB binding site for drought, ABA-responsive, and stress-responsive element. Most of the genes were induced immediately after Magnaporthe oryzae infection at 12 h post-infection (hpi). About 55% of the total genes were upregulated under salt and drought stresses during the entire time, and 22-35% of the total genes were upregulated at 3 h. It was noteworthy that the seven genes (OsCHR705, OsCHR706, OsCHR710, OsCHR714, OsCHR721, OsCHR726, and OsCHR737) were upregulated, and one gene (OsCHR712) was downregulated under salt and drought stresses, respectively. The deficiency of OsCHR726 mutations displayed a hypersensitive phenotype under salt stress. These results will be significantly useful features for the validation of the rice Snf2 genes and facilitate understanding of the genetic engineering of crops with improved biotic and abiotic stresses.

OsDDM1b Controls Grain Size by Influencing Cell Cycling and Regulating Homeostasis and Signaling of Brassinosteroid in Rice.

Snf2 family proteins are the crucial subunits of chromatin-remodeling complexes (CRCs), which contributes to the biological processes of transcription, replication, and DNA repair using ATP as energy. Some CRC subunits have been confirmed to be the critical regulators in various aspects of plant growth and development and in epigenetic mechanisms such as histone modification, DNA methylation, and histone variants. However, the functions of Snf2 family genes in rice were poorly investigated. In this study, the relative expression profile of 40 members of Snf2 family in rice was studied at certain developmental stages of seed. Our results revealed that OsCHR741/OsDDM1b (Decrease in DNA methylation 1) was accumulated highly in the early developmental stage of seeds. We further analyzed the OsDDM1b T-DNA insertion loss-of-function of mutant, which exhibited dwarfism, smaller organ size, and shorter and wider grain size than the wild type (Hwayoung, HY), yet no difference in 1,000-grain weight. Consistent with the grain size, the outer parenchyma cell layers of lemma in osddm1b developed more cells with decreased size. OsDDM1b encoded a nucleus, membrane-localized protein and was distributed predominately in young spikelets and seeds, asserting its role in grain size. Meanwhile, the osddm1b was less sensitive to brassinosteroids (BRs) while the endogenous BR levels increased. We detected changes in the expression levels of the BR signaling pathway and feedback-inhibited genes with and without exogenous BR application, and the alterations of expression were also observed in grain size-related genes in the osddm1b. Altogether, our results suggest that OsDDM1b plays a crucial role in grain size via influencing cell proliferation and regulating BR signaling and homeostasis.

The Interaction between Rice Genotype and Magnaporthe oryzae Regulates the Assembly of Rice Root-Associated Microbiota.

BACKGROUND: Utilizating the plant microbiome to enhance pathogen resistance in crop production is an emerging alternative to the use of chemical pesticides. However, the diversity and structure of the microbiota, and the assembly mechanisms of root-associated microbial communities of plants are still poorly understood. RESULTS: We invstigated the microbiota of the root endosphere and rhizosphere soils of the rice cultivar Nipponbare (NPB) and its Piz-t-transgenic line (NPB-Piz-t) when infected with the filamentous fungus Magnaporthe oryzae (M. oryzae) isolate KJ201, using 16S rRNA and internal transcribed spacer 1 (ITS1) amplicon sequencing. The rhizosphere soils showed higher bacterial and fungal richness and diversity than the endosphere except for fungal richness in the rhizosphere soils of the mock treatment. Bacteria richness and diversity increased in the endospheric communities of NPB and Piz-t under inoculation with KJ201 (referred to as 'NPB-KJ201' and 'Piz-t-KJ201', respectively) compared with the corresponding mock treatments, with the NPB-KJ201 showing the highest diversity in the four bacterial endocompartments. In contrast, fungal richness and diversity decreased in the endospheric communities of NPB-KJ201 and Piz-t-KJ201, relative to the corresponding mock treatments, with NPB-KJ201 and Piz-t-KJ201 having the lowest richness and diversity, respectively, across the four fungal endocompartments. Principal component analysis (PCA) indicated that the microbiota of Piz-t-KJ201 of root endophytes were mostly remarkablely distinct from that of NPB-KJ201. Co-occurrence network analysis revealed that the phyla Proteobacteria and Ascomycota were the key contributors to the bacterial and fungal communities, respectively. Furthermore, a comparative metabolic analysis showed that the contents of tryptophan metabolism and indole alkaloid biosynthesis were significantly lower in the Piz-t-KJ201 plants. CONCLUSIONS: In this study, we compared the diversity, composition, and assembly of microbial communities associated with the rhizosphere soils and endosphere of Piz-t-KJ201 and NPB-KJ201. On the basis of the different compositions, diversities, and assemblies of the microbial communities among different compartments, we propose that the host genotype and inoculation pattern of M. oryzae played dominant roles in determining the microbial community assemblage. Further metabolomics analysis revealed that some metabolites may influence changes in bacterial communities. This study improves our understanding of the complex interactions between rice and M. oryzae, which could be useful in developing new strategies to improve rice resistance through the manipulation of soil microorganisms.

Weighted Gene Co-Expression Network Coupled with a Critical-Time-Point Analysis during Pathogenesis for Predicting the Molecular Mechanism Underlying Blast Resistance in Rice.

BACKGROUND: Rice blast, caused by the ascomycete fungus M. oryzae, is one of the most important diseases of rice. Although many blast resistance (R) genes have been identified and deployed in rice varieties, the molecular mechanisms responsible for the R gene-mediated defense responses are yet not fully understood. RESULTS: In this study, we used comparative transcriptomic analysis to explore the molecular mechanism involved in Piz-t-mediated resistance in a transgenic line containing Piz-t (NPB-Piz-t) compared to Nipponbare (NPB). Clustering and principal component analysis (PCA) revealed that the time-point at 24-h post inoculation (hpi) was the most important factor distinguishing the four time-points, which consisted of four genes of mitogen-activated protein kinases (MAPKs) signaling pathway, one gene related to WRKY DNA-binding domain containing protein, five pathogenesis-related protein (OsPR1s) genes, and three genes of R proteins involving in the most significant protein-protein interaction (PPI) pathway. Using weighted gene co-expression network analysis (WGCNA) to investigate RNA-seq data across 0, 24, 48, and 72 hpi, nine modules with similar patterns expression pattern (SEP) and three modules with differential expression pattern (DEP) between NPB-Piz-t and NPB across 0, 24, 48, and 72 hpi with KJ201 (referred to as Piz-t-KJ201 and NPB-KJ201) were identified. Among these the most representative SEP green-yellow module is associated with photosynthesis, and DEP pink module comprised of two specific expressed nucleotide-binding domain and leucine-rich repeat (NLR) genes of LOC_Os06g17900 and LOC_Os06g17920 of Pi2/9 homologous, three NLR genes of LOC_Os11g11810, LOC_Os11g11770, and LOC_Os11g11920 which are putatively associated with important agronomic traits, and a B3 DNA binding domain containing protein related genes (LOC_Os10g39190). Knockout of LOC_Os10g39190 via CRISPR-Cas9 resulted in plant death at the seedling stage. CONCLUSIONS: The research suggested that Piz-t and multiple NLR network might play important roles in the regulation of the resistance response in the Piz-t-KJ201 interaction system. The identified genes provide an NLR repository to study the rice-M. oryzae interaction system and facilitate the breeding of blast-resistant cultivars in the future.

Functional Identification of Novel Cell Death-inducing Effector Proteins from Magnaporthe oryzae.

BACKGROUND: Secreted effector proteins play critical roles in plant-fungal interactions. The Magnaporthe oryzae genome encodes a large number of secreted proteins. However, the function of majority of M. oryzae secreted proteins remain to be characterized. We previously identified 851 in planta-expressed M. oryzae genes encoding putative secreted proteins, and characterized five M. oryzae cell death-inducing proteins MoCDIP1 to MoCDIP5. In the present study, we expand our work on identification of novel MoCDIP proteins. RESULTS: We performed transient expression assay of 98 more in planta-expressed M. oryzae putative secreted protein genes, and identified eight novel proteins, MoCDIP6 to MoCDIP13, that induced plant cell death. Yeast secretion assay and truncation expression analysis revealed that the signal peptides that led the secretion of proteins to the extracellular space, were required for cell death inducing activity of the novel MoCDIPs except for MoCDIP8. Exogenous treatment of rice seedlings with recombinant MoCDIP6 or MoCDIP7 resulted in enhanced resistance to blast fungus, indicating that the two MoCDIPs trigger cell death and elicit defense responses in rice. CONCLUSIONS: The newly identified MoCDIP6 to MoCDIP13, together with previously identified MoCDIP1 to MoCDIP5, provide valuable targets for further dissection of the molecular mechanisms underlying the rice-blast fungus interaction.

Proteomic analysis of the defense response to Magnaporthe oryzae in rice harboring the blast resistance gene Piz-t.

BACKGROUND: Rice blast (caused by Magnaporthe oryzae) is one of the most destructive diseases of rice. While many blast resistance (R) genes have been identified and deployed in rice cultivars, little is known about the R gene-mediated defense mechanism. We used a rice transgenic line harboring the resistance gene Piz-t to investigate the R gene-mediated resistance response to infection. RESULTS: We conducted comparative proteome profiling of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and wild-type Nipponbare (NPB) inoculated with M. oryzae at 24, 48, 72 h post-inoculation (hpi) using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Comparative analysis of the response of NPB-Piz-t to the avirulent isolate KJ201 and the virulent isolate RB22 identified 114 differentially expressed proteins (DEPs) between KJ201-inoculated NPB-Piz-t (KJ201-Piz-t) and mock-treated NPB-Piz-t (Mock-Piz-t), and 118 DEPs between RB22-inoculated NPB-Piz-t (RB22-Piz-t) and Mock-Piz-t. Among the DEPs, 56 occurred commonly in comparisons KJ201-Piz-t/Mock-Piz-t and RB22-Piz-t/Mock-Piz-t. In a comparison of the responses of NPB and NPB-Piz-t to isolate KJ201, 93 DEPs between KJ201-Piz-t and KJ201-NPB were identified. DEPs in comparisons KJ201-Piz-t/Mock-Piz-t, RB22-Piz-t/Mock-Piz-t and KJ201-Piz-t/KJ201-NPB contained a number of proteins that may be involved in rice response to pathogens, including pathogenesis-related (PR) proteins, hormonal regulation-related proteins, defense and stress response-related proteins, receptor-like kinase, and cytochrome P450. Comparative analysis further identified 7 common DEPs between the comparisons KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t, including alcohol dehydrogenase I, receptor-like protein kinase, endochitinase, similar to rubisco large subunit, NADP-dependent malic enzyme, and two hypothetical proteins. CONCLUSIONS: Our results provide a valuable resource for discovery of complex protein networks involved in the resistance response of rice to blast fungus.

Endosperm-specific OsPYL8 and OsPYL9 act as positive regulators of the ABA signaling pathway in rice seed germination.

Pyrabactin resistance-like (PYL) proteins were identified as receptors of the plant hormone ABA. The PYL family consists of multiple members that are differently expressed in various tissues, exhibit distinct biochemical properties and have diverse biological functions. In the present study, we explored the expression patterns of the rice (Oryza sativa L.) OsPYL family genes and determined that OsPYL8 and OsPYL9 are specifically expressed in the endosperms. Sequence analysis and deletion experiments revealed that the OsPYL8 and OsPYL9 promoters contain multiple motifs involved in endosperm-specific expression. Transgenic rice plants overexpressing OsPYL8 or OsPYL9 showed hypersensitivity to ABA during seed germination, suggesting that both OsPYL8 and OsPYL9 act as positive regulators of the ABA signalling pathway in the seed. OsPYL8 and OsPYL9 interact with OsPP2C51 and OsPP2C68, whose expression is induced in the endosperms by ABA. Our results provided a foundation for future studies on OsPYL8- and OsPYL9-mediated ABA signalling in the rice endosperms.

Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars.

BACKGROUND: The most sustainable approach to control rice blast disease is to develop durably resistant cultivars. In molecular breeding for rice blast resistance, markers developed based on polymorphisms between functional and non-functional alleles of resistance genes, can provide precise and accurate selection of resistant genotypes without the need for difficult, laborious and time-consuming phenotyping. The Pi2 and Pi9 genes confer broad-spectrum resistance against diverse blast isolates. Development of allele-specific markers for Pi2 and Pi9 would facilitate breeding of blast resistant rice by using the two blast resistance genes. RESULT: In this work, we developed two new markers, named Pi9-Pro and Pi2-LRR respectively, targeting the unique polymorphisms of the resistant and susceptible alleles of Pi2 and of Pi9. The InDel marker Pi9-Pro differentiates three different genotypes corresponding to the Pi2/Piz-t, Pi9 and non-Pi2/Piz-t/Pi9 alleles, and the CAPS marker Pi2-LRR differentiates the Pi2 allele from the non-Pi2 allele. Based on the two newly developed markers and two available markers Pi2SNP and Pi9SNP, the presence of Pi2 and Pi9 was assessed in a set of 434 rice accessions consisting of 377 Chinese indica cultivars/breeding materials and 57 Chinese japonica cultivars/breeding materials. Of the 434 accessions tested, while one indica restorer line Huazhan was identified harboring the Pi2 resistance allele, no other rice line was identified harboring the Pi2 or Pi9 resistance alleles. CONCLUSIONS: Allele-specific marker-based assessment revealed that Pi2 and Pi9 have not been widely incorporated into diverse Chinese indica rice cultivars. Thus, the two blast resistance genes can be new gene sources for developing blast resistant rice, especially indica rice, in China. The two newly developed markers should be highly useful for using Pi2 and Pi9 in marker-assisted selection (MAS) breeding programs.

A high-density intervarietal map of the wheat genome enriched with markers derived from expressed sequence tags.

Bread wheat (Triticum aestivum L.) is a hexaploid species with a large and complex genome. A reference genetic marker map, namely the International Triticeae Mapping Initiative (ITMI) map, has been constructed with the recombinant inbred line population derived from a cross involving a synthetic line. But it is not sufficient for a full understanding of the wheat genome under artificial selection without comparing it with intervarietal maps. Using an intervarietal mapping population derived by crossing Nanda2419 and Wangshuibai, we constructed a high-density genetic map of wheat. The total map length was 4,223.1 cM, comprising 887 loci, 345 of which were detected by markers derived from expressed sequence tags (ESTs). Two-thirds of the high marker density blocks were present in interstitial and telomeric regions. The map covered, mostly with the EST-derived markers, approximately 158 cM of telomeric regions absent in the ITMI map. The regions of low marker density were largely conserved among cultivars and between homoeologous subgenomes. The loci showing skewed segregation displayed a clustered distribution along chromosomes and some of the segregation distortion regions (SDR) are conserved in different mapping populations. This map enriched with EST-derived markers is important for structure and function analysis of wheat genome as well as in wheat gene mapping, cloning, and breeding programs.

Molecular genetic analysis of five spike-related traits in wheat using RIL and immortalized F2 populations.

Kernel number per spike is one of the most important yield components of wheat. To map QTLs related to kernel number including spike length (SPL), spikelet number per spike (SPN), fertile spikelet number (FSPN), sterile spikelet number (SSPN) and compactness, and to characterize the inheritance modes of the QTLs and two-locus interactions, 136 recombinant inbred lines (RILs) derived from 'Nanda2419' x 'Wangshuibai' and an immortalized F(2 )population (IF(2)) generated by randomly permutated intermating of these RILs were investigated. QTL mapping made use of the previously constructed over 3300 cM linkage map of the RIL population. Three, five, two, two and six chromosome regions were identified, respectively, for their association with SPL, SPN, FSPN, SSPN, and compactness in at least two of the three environments examined. All compactness QTLs but one shared the respective intervals of QSpn.nau-5A and the SPL QTLs. Xcfd46-Xwmc702 interval on chromosome 7D was related to all traits but SSPN and had consistently the largest effects. The fact that not all the compactness QTL intervals were related to both SPL and SPN indicates that compactness is regulated by different mechanisms. Interval coincidence between QTLs of SPL and SPN and between QTLs of FSPN and SSPN was minimal. For all the traits, favorable alleles exist in both parents. Inheritance modes from additiveness to overdominance of the QTLs were revealed and two-locus interactions were detected, implying that the traits studied are under complex genetic control. The results could contribute to wheat yield improvement and better use of Wangshuibai and Nanda2419 the two special germplasms in wheat breeding program.