Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Cloning, Expression Pattern Analysis and Subcellular Localization of Resveratrol Synthase Gene in Peanut (<i>Arachis hypogaea</i>L.)

Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.

The Intronic cis Element SE1 Recruits trans-Acting Repressor Complexes to Repress the Expression of ELONGATED UPPERMOST INTERNODE1 in Rice

Plant height has a major effect on grain yield in crops such as rice （Oryza sativa）, and the hormone gibberellic acid （GA） regulates many developmental processes that feed into plant height. Rice ELONGATED UPPERMOST INTERNODE1 （Euil） encodes a GA-deactivating enzyme governing elongation of the uppermost internode. The expression of Euil is finely tuned, thereby maintaining homeostasis of endogenous bioactive GA and producing plants of normal plant height. Here, we identified a dominant dwarf mutant, dEuil, caused by the deletion of an RY motif-containing cis-silencing element （SE1） in the intron of Euil. Detailed genetic and molecular analysis of SE1 revealed that this intronic cis element recruits at least one trans-acting repressor complex, containing the B3 repressors OsVAL2 and OsGD1, the SAP18 corepressor, and the histone deacetylase OsHDA710, to negatively regulate the expression of Euil. This com- plex generates closed chromatin at Euil, suppressing Euil expression and modulating GA homeostasis. Loss of SE1 or dysfunction of the complex components impairs histone deacetylation and H3K27me3 methylation of Euil chromatin, thereby increasing Euil transcription and decreasing bioactive GA, producing dwarfism in rice. Together, our results reveal a novel silencing mechanism in which the intronic cis element SE1 negatively regulates Euil expression via repressor complexes that modulate histone deacetylation and/or methylation.

A novel CCCH-type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice

Male sterility is a prerequisite for hybrid seed production.The phytohormone gibberellin(GA)is in-volved in regulating male reproductive development,but the mechanism underlying GA homeostasis in anther development remains less understood.Here,we report the isolation and characterization of a new positive regulator of GA homeostasis,swollen anther wall 1(SAW1),for anther development in rice(Oryza sativa L.).Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants.SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein,and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression.In the saw1 anther,the significantly decreased OsGA20ox3 expression resulted in lower bio-active GA content,which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB.Thus,our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development,and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice

Male sterility(MS),characterized by functional defects in male organs or gametes,is an important agronomic trait for hybrid seed production,especially for self-pollinated crops such as rice(Oryza sativa L)(Chen and Liu,2014).Spontaneous MS mutants are rare and difficult to maintain in nature,thus limiting basicresearch and breeding.Artificial mutants are typically generated by physical,chemical,or biological mutagenesis(Wei et al,2013).Recently developed genome editing systems such as CRISPR/Cas9 allow efficient and timesaving knockout of endogenous genes at specific sites(Smith et al,2000;Moscou and Bogdanove,2009;Gasiunas et al.,2012).

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.

OsEDM2L mediates m^(6)A of EAT1 transcript for proper alternative splicing and polyadenylation regulating rice tapetal degradation

N^(6)-methyladenosine(m^(6)A) modification affects the post-transcriptional regulation of eukaryotic gene expression, but the underlying mechanisms and their effects in plants remain largely unknown. Here,we report that the N^(6)-adenine methyltransferase-like domain-containing protein ENHANCED DOWNY MILDEW 2-LIKE(OsEDM2 L) is essential for rice(Oryza sativa L.) anther development. The osedm2 l knockout mutant showed delayed tapetal programmed cell death(PCD) and defective pollen development. OsEDM2 L interacts with the transcription factors basic helix-loop-helix 142 and TAPETUMDEGENERATIONRETARDATIONto regulate the expression of ETERNAL TAPETUM 1(EAT1), a positive regulator of tapetal PCD. Mutation of OsEDM2 L altered the transcriptomic m^(6)A landscape, and caused a distinct m^(6)A modification of the EAT1 transcript leading to dysregulation of its alternative splicing and polyadenylation, followed by suppression of the EAT1 target genes OsAP25 and OsAP37 for tapetal PCD. Therefore, OsEDM2 L is indispensable for proper messenger RNA m^(6)A modification in rice anther development.

OsLTP47 may function in a lipid transfer relay essential for pollen wall development in rice

In plants,lipid transfer proteins(LTPs)transport pollen wall constituents from the tapetum to the exine,a process essential for pollen wall development.However,the functional cooperation of different LTPs in pollen wall development is not well understood.In this study,we have identified and characterized a grassspecific LTP gene,Os LTP47,an important regulator of pollen wall formation in rice(Oryza sativa).Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays confirms that Os LTP47 has lipidbinding activity.Dysfunction of Os LTP47 causes disordered lipid metabolism and defective pollen walls,leading to male sterility.Yeast two-hybrid and pull-down assays reveal that Os LTP47 physically interacts with another LTP,Os C6.These findings suggest that the plasma membrane-localized Os LTP47 may function as a mediator in a lipid transfer relay through association with cytosolic and/or locular Os C6 for pollen wall development and that various LTPs may function in a coordinated manner to transport lipid molecules during pollen wall development.