A fungal effector and a rice NLR protein have antagonistic effects on a Bowman-Birk trypsin inhibitor.

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.

The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice.

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.

Mild Encephalitis/Encephalopathy with Reversible Splenial Lesion (MERS) due to Cytomegalovirus: Case Report and Review of the Literature.

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.

The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice.

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa.

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.

Genome-Wide Association Mapping of Rice Resistance Genes Against Magnaporthe oryzae Isolates from Four African Countries.

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.

HDT701, a histone H4 deacetylase, negatively regulates plant innate immunity by modulating histone H4 acetylation of defense-related genes in rice.

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.

The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice.

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.

Familial cleidocranial dysplasia misdiagnosed as rickets over three generations.

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.

A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition 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.

Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice.

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.

A young boy with ventricular arrhythmias and thyroid dysgenesis: two genes are not enough?

Congenital hypothyroidism (CH) may be caused by biallelic variants in the TSHR gene. CH due to thyroid dysgenesis has also been linked to pathogenic variants of the nucleotide kinase 2, homeobox 5 (NKX2-5) gene, which can also cause sudden cardiac death from ventricular arrhythmia. In particular, the NKX2-5 p.Arg25Cys missense variant has been repeatedly reported in patients with congenital heart defects and, more rarely, with hypogonadism. We report the case of a 7 year old boy with ventricular arrhythmias, thyroid dysgenesis and intellectual disability, born from consanguineous Tunisian parents. Exome sequencing and segregation analysis revealed two potentially relevant variants: the NKX2-5 p.Arg25Cys variant (maternally inherited), as well as a single heterozygous TSHR p.Gln90Pro variant (paternally inherited). Of note, a male sibling of the proband, presenting with intellectual disability only, carried the same two variants. No other TSHR variants, or other potentially relevant variants were identified. In this proband, despite the identification of variants in two genes potentially correlated to the phenotype, a definite genetic diagnosis could not be reached. This case report highlights the complexity of exome data interpretation, especially when dealing with families presenting complex phenotypes and variable expression of clinical traits.

Seronegative phenotype in a pediatric population with Hashimoto's thyroiditis.

PURPOSE: 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. SUBJECTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Clinical and Brain Imaging Findings in a Child with Vitamin B12 Deficiency.

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.

Cycling for a Sustainable Touristic Mobility: A Preliminary Study in an Urban Area of Italy.

This study wants to give a contribution for the investigation of sustainable mobility with positive consequences on public health implementing policies starting from cyclists' perceptions. Data were collected by interviewing cyclists along three bike lanes of an urban area of southern Italy through a face-to-face survey. The survey was conducted in Autumn 2019, interviewing a sample of 129 cyclists. In order to identify the critical aspects of the bike paths, both an importance-performance analysis (IPA) and a gap-IPA were performed. The average values of the cyclists' perceptions of each aspect have been considered as performance values. The importance values have been obtained by performing a principal component analysis (PCA), which was helpful also for better defining the service quality phenomenon. From the PCA, six latent constructs can be identified as: "Physical Nuisance"; "Non-physical Nuisance"; "Physical Comfort"; "Non-physical Comfort"; "Protection"; and "Ambience". The results of Gap-IPA confirmed that the criticalities of the bike paths relate to the degree of protection in relation to accidents, and to the degree of nuisance caused by pollution and opposing pedestrians along the path. Based on the conducted analyses, sustainable tourism implementing policies should be oriented in solving the emerged criticalities of the existing bike paths. The results of Gap-IPA are very intuitive and can certainly be helpful for identifying the most convenient strategies.

Generalized Arterial Calcification of Infancy Type 1 (GACI1): Identification of a Novel Pathogenic Variant (c.1715T>C (p.Leu572Ser)).

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.

Two VOZ transcription factors link an E3 ligase and an NLR immune receptor to modulate immunity in rice.

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.

A CRISPR/dCas9 toolkit for functional analysis of maize genes.

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.

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

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

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity.

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.