SPOTTED-LEAF7 targets the gene encoding ß-galactosidase9, which functions in rice growth and stress responses.

ß-Galactosidases (Bgals) remove terminal ß-D-galactosyl residues from the nonreducing ends of ß-D-galactosidases and oligosaccharides. Bgals are present in bacteria, fungi, animals, and plants and have various functions. Despite the many studies on the evolution of BGALs in plants, their functions remain obscure. Here, we identified rice (Oryza sativa) ß-galactosidase9 (OsBGAL9) as a direct target of the heat stress-induced transcription factor SPOTTED-LEAF7 (OsSPL7), as demonstrated by protoplast transactivation analysis and yeast 1-hybrid and electrophoretic mobility shift assays. Knockout plants for OsBGAL9 (Osbgal9) showed short stature and growth retardation. Histochemical ß-glucuronidase (GUS) analysis of transgenic lines harboring an OsBGAL9pro:GUS reporter construct revealed that OsBGAL9 is mainly expressed in internodes at the mature stage. OsBGAL9 expression was barely detectable in seedlings under normal conditions but increased in response to biotic and abiotic stresses. Ectopic expression of OsBGAL9 enhanced resistance to the rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, as well as tolerance to cold and heat stress, while Osbgal9 mutant plants showed the opposite phenotypes. OsBGAL9 localized to the cell wall, suggesting that OsBGAL9 and its plant putative orthologs likely evolved functions distinct from those of its closely related animal enzymes. Enzyme activity assays and analysis of the cell wall composition of OsBGAL9 overexpression and mutant plants indicated that OsBGAL9 has activity toward galactose residues of arabinogalactan proteins (AGPs). Our study clearly demonstrates a role for a member of the BGAL family in AGP processing during plant development and stress responses.

Sucrose preferentially promotes expression of OsWRKY7 and OsPR10a to enhance defense response to blast fungus in rice.

Sucrose controls various developmental and metabolic processes in plants. It also functions as a signaling molecule in the synthesis of carbohydrates, storage proteins, and anthocyanins, as well as in floral induction and defense response. We found that sucrose preferentially induced OsWRKY7, whereas other sugars (such as mannitol, glucose, fructose, galactose, and maltose) did not have the same effect. A hexokinase inhibitor mannoheptulose did not block the effect of sucrose, which is consequently thought to function directly. MG132 inhibited sucrose induction, suggesting that a repressor upstream of OsWRKY7 is degraded by the 26S proteasome pathway. The 3-kb promoter sequence of OsWRKY7 was preferentially induced by sucrose in the luciferase system. Knockout mutants of OsWRKY7 were more sensitive to the rice blast fungus Magnaporthe oryzae, whereas the overexpression of OsWRKY7 enhanced the resistance, indicating that this gene is a positive regulator in the plant defense against this pathogen. The luciferase activity driven by the OsPR10a promoter was induced by OsWRKY7 and this transcription factor bound to the promoter region of OsPR10a, suggesting that OsWRKY7 directly controls the expression of OsPR10a. We conclude that sucrose promotes the transcript level of OsWRKY7, thereby increasing the expression of OsPR10a for the defense response in rice.

Heat stress transcription factor OsSPL7 plays a critical role in reactive oxygen species balance and stress responses in rice.

The rice spotted leaf gene, OsSPL7, induces lesion mimic (LM) spots under heat stress. Herein, we provide several lines of evidence elucidating the importance of OsSPL7 in maintaining reactive oxygen species (ROS) balance via the regulation of downstream gene expression. osspl7 knockout (spl7ko) mutants showed LM and growth retardation. Transgenic rice lines strongly overexpressing OsSPL7 (SPL7OX-S) exhibited LM accompanied by accumulated H2O2, whereas moderate expressers of OsSPL7 (SPL7OX-M) did not, and neither of them exhibited severe growth defects. Transient expression of OsSPL7-GFP in rice protoplasts indicated that OsSPL7 localizes predominantly in the nucleus. Transcriptional activity assay suggested its function as a transcriptional activator in rice. Disease evaluation showed that both SPL7OX and spl7ko enhanced resistance to Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae, the causal agents of blast and blight diseases in rice, respectively. Additionally, SPL7OX enhanced tolerance to cold stress, whereas spl7ko showed a phenotype opposite to the overexpression lines. RNA sequencing analyses identified four major groups of differentially expressed genes associated with LM, pathogen resistance, LM-pathogen resistance, and potential direct targets of OsSPL7. Collectively, our results suggest that OsSPL7 plays a critical role in plant growth and balancing ROS during biotic and abiotic stress.

Pi5 and Pii Paired NLRs Are Functionally Exchangeable and Confer Similar Disease Resistance Specificity.

Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii -carrying cultivar Fujisaka5 . The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii , similar to Pi5 , requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2 . Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2 , respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.