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Nucleotide-binding leucine-rich repeat (NLR) proteins play critical roles in plant immunity. However, how NLRs are regulated and activate defense signaling is not fully understood. The rice (Oryza sativa) NLR receptor Piz-t confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae and the RING-type E3 ligase AVRPIZ-T INTERACTING PROTEIN 10 (APIP10) negatively regulates Piz-t accumulation. In this study, we found that APIP10 interacts with two rice transcription factors, VASCULAR PLANT ONE-ZINC FINGER 1 (OsVOZ1) and OsVOZ2, and promotes their degradation through the 26S proteasome pathway. OsVOZ1 displays transcriptional repression activity while OsVOZ2 confers transcriptional activation activity in planta. The osvoz1 and osvoz2 single mutants display modest but opposite M. oryzae resistance in the non-Piz-t background. However, the osvoz1 osvoz2 double mutant exhibits strong dwarfism and cell death, and silencing of both genes via RNA interference also leads to dwarfism, mild cell death, and enhanced resistance to M. oryzae in the non-Piz-t background. Both OsVOZ1 and OsVOZ2 interact with Piz-t. Double silencing of OsVOZ1 and OsVOZ2 in the Piz-t background decreases Piz-t protein accumulation and transcription, reactive oxygen species-dependent cell death, and resistance to M. oryzae containing AvrPiz-t. Taken together, these results indicate that OsVOZ1 and OsVOZ2 negatively regulate basal defense but contribute positively to Piz-t-mediated immunity.

Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease.

Summary Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz‐t targets the host ubiquitin‐proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz‐t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz‐t, we generated double (K1,2R‐AvrPiz‐t), triple (K1,2,3R‐AvrPiz‐t) and lysine‐free (LF‐AvrPiz‐t) mutants by mutating lysines into arginines in AvrPiz‐t. LF‐AvrPiz‐t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co‐expressed with APIP10 in Nicotiana benthamiana, LF‐AvrPiz‐t was more stable than AvrPiz‐t and was less able to degrade APIP10. The avirulence of LF‐AvrPiz‐t on Piz‐t:HA plants was less than that of AvrPiz‐t, which led to resistance reduction and lower accumulation of the Piz‐t:HA protein after inoculation with the LF‐AvrPiz‐t‐carrying isolate. Chitin‐ and flg22‐induced production of reactive oxygen species (ROS) was higher in LF‐AvrPiz‐t than in AvrPiz‐t transgenic plants. In addition, LF‐AvrPiz‐t transgenic plants were less susceptible than AvrPiz‐t transgenic plants to a virulent isolate. Furthermore, both AvrPiz‐t and LF‐AvrPiz‐t interacted with OsRac1, but the suppression of OsRac1‐mediated ROS generation by LF‐AvrPiz‐t was significantly lower than that by AvrPiz‐t. Together, these results suggest that the lysine residues of AvrPiz‐t are required for its avirulence and virulence functions in rice.

The aim was to verify in a pediatric population with Hashimoto's thyroiditis whether there is a relationship between antithyroid antibodies and inflammatory status on thyroid ultrasound and thyroid function.A total of 154 children and adolescents, aged 4 to 18 years, diagnosed with Hashimoto's thyroiditis with normal body weight were followed up for 1 year.Patients with only antiperoxidase antibodies presented with higher TSH levels than subjects with only antithyroglobulin antibodies (p 0.027) but with similar FT4 levels and thyroid score. Prevalence of seronegative Hashimoto's thyroiditis in this cohort was 12.3% (19/154). At diagnosis, the seronegative group presented with lower prevalence of overt hypothyroidism, symptoms of hypothyroidism, and thyroid score, meaning less severe thyroid involvement. In contrast, similar TSH and FT4 values were found at diagnosis and during follow-up in both the seronegative and seropositive groups. A comparison between patients with seronegative Hashimoto's thyroiditis and an overweight/obese antibody-negative population, who presented superimposable altered parenchymal pattern on thyroid ultrasound without circulating antithyroid antibodies, presented similar clinical data.We report for the first time in the literature that seronegative Hashimoto's thyroiditis in the pediatric age group has a less severe pattern. The seronegative group presents similar characteristics to those of overweight/obese children and adolescents with ultrasound changes, but, according to the established knowledge, the latter condition is reversible and does not need follow-up examinations.

Vitamin B12 (Vit-B12) deficiency is a rare and treatable cause of failure to thrive and delayed development in infants who are exclusively breastfed. Apart from genetic causes, it can be related to a malabsorption syndrome or when the mother follows a strict vegetarian or vegan diet, causing a low hepatic storage of Vit-B12 in the infant at birth. As the neurological symptoms are nonspecific, a brain magnetic resonance imaging (MRI) exam is usually performed to rule out primary causes of neurodevelopmental delay. Findings related to brain atrophy are usually observed. A favorable response is achieved with Vit-B12 therapy, and neurological symptoms dramatically improve within a few days after the treatment. We present the case of an infant with severe Vit-B12 deficiency, exclusively breastfed by his young vegan mother, and whose clinical symptoms together with MRI findings improved after treatment. Brain atrophy recovery after Vit-B12 therapy has been seldom documented.

Summary Bowman–Birk trypsin inhibitors (BBIs) play important roles in animal and plant immunity, but how these protease inhibitors are involved in the immune system remains unclear. Here, we show that the rice (Oryza sativa) BBI protein APIP4 is a common target of a fungal effector and an NLR receptor for innate immunity. APIP4 exhibited trypsin inhibitor activity in vitro and in vivo. Knockout of APIP4 in rice enhanced susceptibility, and overexpression of APIP4 increased resistance to the fungal pathogen Magnaporthe oryzae. The M. oryzae effector AvrPiz‐t interacted with APIP4 and suppressed APIP4 trypsin inhibitor activity. By contrast, the rice NLR protein Piz‐t interacted with APIP4, enhancing APIP4 transcript and protein levels, and protease inhibitor activity. Our findings reveal a novel host defence mechanism in which a host protease inhibitor targeted by a fungal pathogen is protected by an NLR receptor.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Mild encephalopathy with a reversible splenial lesion (MERS) is a clinico-radiological syndrome characterized by a transient mild encephalopathy and MRI findings of a reversible lesion in the splenium of corpus callosum (SCC). It is classified in MERS type I and MERS type II, depending on the involvement of SCC alone or also other white matter areas. The syndrome mainly affects children and young adults; the prognosis is favorable with complete or nearly complete neurological and radiological resolution within days or weeks. The vast majority of the cases described in the literature involve Asian and Australian children. The exact pathophysiology is unknown; however, infectious-related MERS (in particular virus associated MERS) remains the most common cause of reversible splenial lesions in childhood. To the best of our knowledge, there is only one published case of MERS associated with cytomegalovirus (CMV) infection involving an Australian child. We present here the first case of a CMV-related MERS in a European Caucasian child.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia characterized by hypoplastic clavicles, late closure of the fontanels, dental problems and other skeletal features. CCD is caused by mutations, deletions or duplications in runt-related transcription factor 2 (RUNX2), which encodes for a protein essential for osteoblast differentiation and chondrocyte maturation. We describe three familial cases of CCD, misdiagnosed as rickets over three generations. No mutations were detected on standard DNA sequencing of RUNX2, but a novel deletion was identified on quantitative polymerase chain reaction (qPCR) and multiple ligation-dependent probe amplification (MLPA). The present cases indicate that CCD could be misdiagnosed as rickets, leading to inappropriate treatment, and confirm that mutations in RUNX2 are not able to be identified on standard DNA sequencing in all CCD patients, but can be identified on qPCR and MLPA.

Rice, as a major crop in the world, produces huge agronomic biomass residues besides food, which consist of cellulose, hemicelluloses and lignin. Many master regulators of secondary wall synthesis were identified in the model plant Arabidopsis. In this study, we investigated the function of a NAC (NAM, ATAF, and CUC2) transcription factor related to secondary cell wall biosynthesis, which is highly expressed in rice sclerenchyma tissue and is named OsSWN1. Our results showed that engineering of OsSWN1 could exhibit multiple features regulated to agronomic traits and bioenergy research. Over-expression of OsSWN1 caused an erect-leaf and enclosed-flower phenotype. Secondary cell wall-related genes were actively expressed in transgenic plants with obvious ectopic lignin deposition in the leaf collar, while increased lignin content and decreased the sugar yield correspondingly. In addition, down-regulation of OsSWN1 expression levels decreased lignin content and increased the sugar yield in transgenic plants. Bioinformatics analysis revealed that OsSWN1-like genes are highly conserved in switchgrass and sorghum, suggesting a possibility of manipulating the expression level of the OsSWN1 orthologs in the bioenergy crops for biofuel production.

Molecular mapping of new blast resistance genes is important for developing resistant rice cultivars using marker-assisted selection. In this study, 259 recombinant inbred lines (RILs) were developed from a cross between Nipponbare and 93-11, and were used to construct a 1165.8-cM linkage map with 131 polymorphic simple sequence repeat (SSR) markers. Four major quantitative trait loci (QTLs) for resistance to six isolates of Magnaporthe oryzae were identified: qPi93-1, qPi93-2, qPi93-3, and qPiN-1. For the three genes identified in 93-11, qPi93-1 is linked with SSR marker RM116 on the short arm of chromosome 11 and explains 33% of the phenotypic variation in resistance to isolate CHE86. qPi93-2 is linked with SSR marker RM224 on the long arm of chromosome 11 and accounts for 31% and 25% of the phenotypic variation in resistance to isolates 162-8B and ARB50, respectively. qPi93-3 is linked with SSR marker RM7102 on chromosome 12 and explains 16%, 53%, and 28% of the phenotypic variation in resistance to isolates CHE86, ARB52, and ARB94, respectively. QTL qPiN-1 from Nipponbare is associated with SSR marker RM302 on chromosome 1 and accounts for 34% of the phenotypic variation in resistance to isolate PO6-6. These new genes can be used to develop new varieties with blast resistance via marker-aided selection and to explore the molecular mechanism of rice blast resistance.

Rice blast disease is emerging as a major constraint to rice production in Africa. Although a traditional gene-tagging strategy using biparental crosses can effectively identify resistance (R) genes or quantitative trait loci (QTL) against Magnaporthe oryzae, the mapping procedure required is time consuming and requires many populations to investigate the genetics of resistance. In this report, we conducted a genome-wide association study (GWAS) to rapidly map rice genes conferring resistance against eight M. oryzae isolates from four African countries. We inoculated 162 rice cultivars, which were part of the rice diversity panel 1 (RDP1) and were previously genotyped with the 44,000 single-nucleotide polymorphism (SNP) chip, with the eight isolates. The GWAS identified 31 genomic regions associated with blast resistance (RABR) in the rice genome. In addition, we used polymerase chain reaction analysis to confirm the association between the Pish gene and a major RABR on chromosome 1 that was associated with resistance to four M. oryzae isolates. Our study has demonstrated the power of GWAS for the rapid identification of rice blast R or QTL genes that are effective against African populations of M. oryzae. The identified SNP markers associated with RABR can be used in breeding for resistance against rice blast in Africa.

Hemibiotrophic pathogens are some of the most destructive plant pathogens, causing huge economic losses and threatening global food security. Infection with these organisms often involves an initial biotrophic infection phase, during which the pathogen spreads in host tissue asymptomatically, followed by a necrotrophic phase, during which host-cell death is induced. How hemibiotrophic pathogens trigger host necrosis and how plants inhibit the transition from the biotrophic stage to the necrotrophic stage in disease symptom expression are mainly unknown. The rice blast fungus Magnaporthe oryzae spreads in rice biotrophically early during infection, but this biotrophic stage is followed by a pronounced switch to cell death and lesion formation. Here, we show that the M. oryzae effector AvrPiz-t interacts with the bZIP-type transcription factor APIP5 in the cytoplasm and suppresses its transcriptional activity and protein accumulation at the necrotrophic stage. Silencing of APIP5 in transgenic rice leads to cell death, and the phenotype is enhanced by the expression of AvrPiz-t. Conversely, Piz-t interacts with and stabilizes APIP5 to prevent necrosis at the necrotrophic stage. At the same time, APIP5 is essential for Piz-t stability. These results demonstrate a novel mechanism for the suppression of effector-triggered necrosis at the necrotrophic stage by an NLR receptor in plants.

Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from Magnaporthe oryzae targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of APIP10 in the non-Piz-t background compromises the basal defense against M. oryzae. Conversely, silencing of APIP10 in the Piz-t background causes cell death, significant accumulation of Piz-t, and enhanced resistance to M. oryzae, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during M. oryzae infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice., Author Summary Rice is the staple food for half of the world’s population. Rice diseases are, however, the major threat for stable rice production worldwide. Elucidating the molecular basis is pivotal for the development of durable resistance to control rice diseases. We previously found that the RING finger E3 ligase APIP6 interacts with AvrPiz-t and plays a role in rice PAMP-triggered immunity (PTI). In this study, we characterized another RING finger E3 ligase in rice, named APIP10. Like APIP6, APIP10 and AvrPiz-t degrade each other, and APIP10 is a positive regulator of PTI. Interestingly, reduction of APIP10 expression level in the Piz-t resistant plants causes severe cell death and accumulation of the NLR receptor Piz-t, indicating APIP10 is a negative regulator of Piz-t. We also show that APIP10 can promote Piz-t degradation while AvrPiz-t can stabilize Piz-t. Our results demonstrate that APIP10 is a target of a fungal effector and a negative regulator of an NLR receptor in plants.

Although the functions of a few effector proteins produced by bacterial and oomycete plant pathogens have been elucidated in recent years, information for the vast majority of pathogen effectors is still lacking, particularly for those of plant-pathogenic fungi. Here, we show that the avirulence effector AvrPiz-t from the rice blast fungus Magnaporthe oryzae preferentially accumulates in the specialized structure called the biotrophic interfacial complex and is then translocated into rice (Oryza sativa) cells. Ectopic expression of AvrPiz-t in transgenic rice suppresses the flg22- and chitin-induced generation of reactive oxygen species (ROS) and enhances susceptibility to M. oryzae, indicating that AvrPiz-t functions to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Interaction assays show that AvrPiz-t suppresses the ubiquitin ligase activity of the rice RING E3 ubiquitin ligase APIP6 and that, in return, APIP6 ubiquitinates AvrPiz-t in vitro. Interestingly, agroinfection assays reveal that AvrPiz-t and AvrPiz-t Interacting Protein 6 (APIP6) are both degraded when coexpressed in Nicotiana benthamiana. Silencing of APIP6 in transgenic rice leads to a significant reduction of flg22-induced ROS generation, suppression of defense-related gene expression, and enhanced susceptibility of rice plants to M. oryzae. Taken together, our results reveal a mechanism in which a fungal effector targets the host ubiquitin proteasome system for the suppression of PAMP-triggered immunity in plants.

Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell. The atypical resistance (R) protein RPW8.2 activates salicylic acid (SA)-dependent, haustorium-targeted defenses against Golovinomyces spp., the causal agents of powdery mildew diseases on multiple plant species. How RPW8.2 activates defense remains uncharacterized. Here, we report that RPW8.2 interacts with the phytochrome-associated protein phosphatase type 2C (PAPP2C) in yeast and in planta as evidenced by co-immunoprecipitation and bimolecular fluorescence complementation assays. Down-regulation of PAPP2C by RNA interference (RNAi) in Col-0 plants lacking RPW8.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway. Moreover, down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death, which correlates with elevated RPW8.2 expression. We further demonstrate that hemagglutinin (HA)-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity. In addition, silencing a rice gene (Os04g0452000) homologous to PAPP2C also results in spontaneous cell death in rice. Combined, our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.

Programmed cell death (PCD) plays critical roles in plant immunity but must be regulated to prevent excessive damage. The E3 ubiquitin ligase SPL11 negatively regulates PCD and immunity in plants. We show that SPL11 cell-death suppressor 2 (SDS2), an S-domain receptor-like kinase, positively regulates PCD and immunity in rice by engaging and regulating SPL11 and related kinases controlling defense responses. An sds2 mutant shows reduced immune responses and enhanced susceptibility to the blast fungus Magnaporthe oryzae. Conversely, SDS2 over-expression induces constitutive PCD accompanied by elevated immune responses and enhanced resistance to M. oryzae. SDS2 interacts with and phosphorylates SPL11, which in turn ubiquitinates SDS2, leading to its degradation. In addition, SDS2 interacts with related receptor-like cytoplasmic kinases, OsRLCK118/176, that positively regulate immunity by phosphorylating the NADPH oxidase OsRbohB to stimulate ROS production. Thus, a plasma membrane-resident protein complex consisting of SDS2, SPL11, and OsRLCK118/176 controls PCD and immunity in rice.

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

The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid–dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced (∼53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages.

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

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

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia characterized by hypoplastic clavicles, late closure of the fontanels, dental problems and other skeletal features. CCD is caused by mutations, deletions or duplications in runt-related transcription factor 2 (RUNX2), which encodes for a protein essential for osteoblast differentiation and chondrocyte maturation. We describe three familial cases of CCD, misdiagnosed as rickets over three generations. No mutations were detected on standard DNA sequencing of RUNX2, but a novel deletion was identified on quantitative polymerase chain reaction (qPCR) and multiple ligation-dependent probe amplification (MLPA). The present cases indicate that CCD could be misdiagnosed as rickets, leading to inappropriate treatment, and confirm that mutations in RUNX2 are not able to be identified on standard DNA sequencing in all CCD patients, but can be identified on qPCR and MLPA.

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta–expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta–expressed cell death–inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

Histone acetylation and deacetylation play an important role in the modification of chromatin structure and regulation of gene expression in eukaryotes. Chromatin acetylation status is modulated antagonistically by histone acetyltransferases and histone deacetylases (HDACs). In this study, we characterized the function of histone deacetylase701 (HDT701), a member of the plant-specific HD2 subfamily of HDACs, in rice (Oryza sativa) innate immunity. Transcription of HDT701 is increased in the compatible reaction and decreased in the incompatible reaction after infection by the fungal pathogen Magnaporthe oryzae. Overexpression of HDT701 in transgenic rice leads to decreased levels of histone H4 acetylation and enhanced susceptibility to the rice pathogens M. oryzae and Xanthomonas oryzae pv oryzae (Xoo). By contrast, silencing of HDT701 in transgenic rice causes elevated levels of histone H4 acetylation and elevated transcription of pattern recognition receptor (PRR) and defense-related genes, increased generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, as well as enhanced resistance to both M. oryzae and Xoo. We also found that HDT701 can bind to defense-related genes to regulate their expression. Taken together, these results demonstrate that HDT701 negatively regulates innate immunity by modulating the levels of histone H4 acetylation of PRR and defense-related genes in rice.

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis , 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17 . Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

BACKGROUND: Intracranial haemorrhage represents the most feared stroke subtype. AIM: To evaluate the burden of intracranial haemorrhage in Tuscany hospitals with special reference to Livorno district. MATERIALS AND METHODS: Data of patients discharged in 2009 from Tuscan and Livorno hospitals with codes ICD-9-CM related to any type of spontaneous intracranial haemorrhage were selected and analyzed. RESULTS: 3,472 patients were discharged from Tuscan hospitals with these diagnoses. Overall mortality was 24.3%. 50% of patients were admitted in Internal Medicine wards. Incidence of intracranial haemorrhage and intracerebral haemorrhage (ICH) in population of Livorno district was 64 and 45/100,000 inhabitants/year with related mortality of 36.5% and 39.4%respectively. Intra-hospital mortality of patients admitted in Livorno hospitals for intracranial haemorrhage were 36.7%. 40% of deaths occurred in the first 48 hours. 69.6% of intracranial haemorrhage were ICHs, 16.8% subaracnoideal. Intra-hospital mortality, admissions for intracranial haemorrhage in respect of total admissions and mortality for intracranial haemorrhage in respect to total mortality increased in the last decade. 23% of patients with intracranial haemorrhage and 16% of patients with ICH underwent to surgical procedures. ICHs related to antithrombotic treatment significantly increased in the last years. Mortality in patients on antithrombotic drugs was three times over compared to that in patients not undergone these drugs (43.7% vs 12.8%, p < 0.01). CONCLUSION: There is an increasing trend in frequency, mortality and hospital burden of intracranial haemorrhage and ICH. Efforts aimed at reducing the burden and consequences of this devasting disease are warranted.

Objective and Importance despite recent progress in imaging, it is still difficult to distinguish between pituitary adenoma and hyperplasia, even using Magnetic Resonance Imaging (MRI) with gadolinium injection. We describe an example of reactive pituitary hyperplasia from primary hypothyroidism that mimicked a pituitary macroadenoma in a child. Clinical Presentation a 10 year old boy presented with headache and statural growth arrest. MRI revealed an intrasellar and suprasellar pituitary mass. Endocrine evaluation revealed primary hypothyroidism. Intervention the patient was started on levothyroxine with resolution of the mass effect. Conclusion primary hypothyroidism should be considered in the differential diagnosis of solid mass lesions of the pituitary gland. Examination of thyroid function in patients with sellar and suprasellar masses revealed by MRI may avoid unnecessary operations which can cause irreversible complications.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. To elucidate the function of effector proteins from M. oryzae in pathogenesis and interaction with the host, we have performed RL-SAGE and MPSS approaches to study the gene expression profiles of M. oryzae during the interaction. The RL-SAGE and MPSS analyses identified 3,441 and 3,004 annotated M. oryzae genes from blast infected rice leaf tissues, respectively. Among them, 217 genes encoding putative secreted proteins, which may play important roles as effectors, were identified. We developed a highly efficient transient protoplast system for gene functional analysis in rice. We present here the detailed procedure of the rice protoplast transient expression system and show the examples of using the system for functional analysis of putative effectors from M. oryzae. The combination of RL-SAGE/MPSS genomic profiling approaches with a protoplast functional assay system has provided an efficient approach for large-scale isolation and analysis of effectors from M. oryzae.

The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1 was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.