Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

The rare sugar D-allose acts as a triggering molecule of rice defence via ROS generation.

Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice.

Jasmonic acid and salicylic acid activate a common defense system in rice.

Jasmonic acid (JA) and salicylic acid (SA) play important roles in plant defense systems. JA and SA signaling pathways interact antagonistically in dicotyledonous plants, but, the status of crosstalk between JA and SA signaling is unknown in monocots. Our rice microarray analysis showed that more than half of the genes upregulated by the SA analog BTH are also upregulated by JA, suggesting that a major portion of the SA-upregulated genes are regulated by JA-dependent signaling in rice. A common defense system that is activated by both JA and SA is thus proposed which plays an important role in pathogen defense responses in rice.

Phosphorylation of D-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L.

We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition.

Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice.

The plant hormone jasmonic acid (JA) has a crucial role in both host immunity and development in plants. Here, we report the importance of JA signaling in the defense system of rice. Exogenous application of JA conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice. Expression of OsJAZ8, a rice jasmonate ZIM-domain protein, was highly up-regulated by JA. OsJAZ8 interacted with a putative OsCOI1, which is a component of the SCF(COI1) E3 ubiquitin ligase complex, in a coronatine-dependent manner. OsJAZ8 also formed heterodimers with other OsJAZ proteins but did not form homodimer. JA treatment caused OsJAZ8 degradation and this degradation was dependent on the 26S proteasome pathway. Furthermore, the JA-dependent OsJAZ8 degradation was mediated by the Jas domain. Transgenic rice plants overexpressing OsJAZ8ΔC, which lacks the Jas domain, exhibited a JA-insensitive phenotype. A large-scale analysis using a rice DNA microarray revealed that overexpression of OsJAZ8ΔC altered the expression of JA-responsive genes, including defense-related genes, in rice. Furthermore, OsJAZ8ΔC negatively regulated the JA-induced resistance to Xoo in rice. On the basis of these data, we conclude that JA plays an important role in resistance to Xoo, and OsJAZ8 acts as a repressor of JA signaling in rice.

Rare sugar D-allose suppresses gibberellin signaling through hexokinase-dependent pathway in Oryza sativa L.

One of the rare sugars, D-allose, which is the epimer of D-glucose at C3, has an inhibitory effect on rice growth, but the molecular mechanisms of the growth inhibition by D-allose were unknown. The growth inhibition caused by D-allose was prevented by treatment with hexokinase inhibitors, D-mannoheptulose and N-acetyl-D-glucosamine. Furthermore, the Arabidopsis glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1, showed a D-allose-insensitive phenotype. D-Allose strongly inhibited the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half rice seeds. The growth of the slender rice1 (slr1) mutant, which exhibits a constitutive gibberellin-responsive phenotype, was also inhibited by D-allose, and the growth inhibition of the slr1 mutant by D-allose was also prevented by D-mannoheptulose treatment. The expressions of gibberellin-responsive genes were down-regulated by D-allose treatment, and the down-regulations of gibberellin-responsive genes were also prevented by D-mannoheptulose treatment. These findings reveal that D-allose inhibits the gibberellin-signaling through a hexokinase-dependent pathway.

D-Psicose induces upregulation of defense-related genes and resistance in rice against bacterial blight.

We examined rice responses to a rare sugar, d-psicose. Rice growth was inhibited by d-psicose but not by common sugars. Microarray analysis revealed that d-psicose treatment caused an upregulation of many defense-related genes in rice, and dose-dependent upregulation of these genes was confirmed by quantitative reverse-transcription polymerase chain reaction. The level of upregulation of defense-related genes by d-psicose was low compared with that by d-allose, which is another rare sugar known to confer induction of resistance to rice bacterial blight in rice. Treatment with d-psicose conferred resistance to bacterial blight in rice in a dose-dependent manner, and the results indicate that d-psicose might be a candidate plant activator for reducing disease development in rice.

A rare sugar, d-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa.

We investigated responses of rice plant to three rare sugars, d-altrose, d-sorbose, and d-allose, due to establishment of mass production methods for these rare sugars. Root growth and shoot growth were significantly inhibited by d-allose but not by the other rare sugars. A large-scale gene expression analysis using a rice microarray revealed that d-allose treatment causes a high upregulation of many defense-related, pathogenesis-related (PR) protein genes in rice. The PR protein genes were not upregulated by other rare sugars. Furthermore, d-allose treatment of rice plants conferred limited resistance of the rice against the pathogen Xanthomonas oryzae pv. oryzae but the other tested sugars did not. These results indicate that d-allose has a growth inhibitory effect but might prove to be a candidate elicitor for reducing disease development in rice.