Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA⁺-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H⁺-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H⁺-ATPase complex, thus reducing proton pump activity to close stomata.

Global warming and emerging plant diseases challenge agricultural food/feed production. We identify mechanism(s) regulating both plant thermotolerance and disease resistance. Using virus-induced gene silencing (VIGS)-based genetic screening, we identify a thioredoxin-like 1 (TRXL1) gene involved in plant nonhost disease resistance and thermotolerance. TRXL1 is reduced, partly degraded via proteases and proteasome, and alters its chloroplast localization during heat stress. TRXL1 interacts with more than 400 proteins, including chaperonin CPN60A, caseinolytic protease (CLPC1), and NADP-dependent malate dehydrogenase (NADP-MDH). Chaperonin 60A (CPN60A) guards TRXL1 from degradation, whereas CLPC1 degrades TRXL1 during heat stress. TRXL1 regulates NADP-MDH activity, leading to an increase in malate level and inhibition of superoxide radical formation. We show that CPN60A and NADP-MDH positively regulate nonhost resistance, and CPN60A positively and CLPC1 negatively regulate thermotolerance. This study shows an antagonistic post-translational regulation of TRXL1 by CPN60A and CLPC1 and regulation of MDH by TRXL1, leading to plant disease resistance and thermotolerance.

As a major adverse environmental factor in most parts of the world, drought causes substantial crop yield losses. Rice (Oryza sativa) is one of the staple foods for more than one-half of the world’s population. Rice plants are sensitive to even mild drought stress and need almost twice the amount of water compared to wheat (Triticum aestivum) or maize (Zea mays). Arabidopsis (Arabidopsis thaliana) small GTPase Nucleolar GTP-binding protein 1 (AtNOG1) plays a role in biotic stress tolerance. Here, we created transgenic rice lines constitutively overexpressing AtNOG1-1 or AtNOG1-2. We also developed rice RNA interference (RNAi) lines that show downregulation of OsNOG1. AtNOG1-1 and AtNOG1-2 overexpressors showed enhanced drought tolerance without compromising grain yield, whereas OsNOG1-RNAi was more susceptible to drought when compared to wild-type plants. Analysis of physiological parameters showed increased cell sap osmolality, relative water content, and abscisic acid (ABA) level, but decreased leaf water loss in AtNOG1-1 or AtNOG1-2 overexpressor lines compared to the control. We found upregulation of several genes involved in ABA and jasmonic acid (JA) signaling, stomata regulation, osmotic potential maintenance, stress protection, and disease resistance in AtNOG1-1 and AtNOG1-2 overexpressor lines compared to the control. We elucidated the role of NOG1-2 and NOG1-1 in regulation of silica body formation around stomata to prevent transpirational water loss. These results provide an avenue to confer drought tolerance in rice.

This protocol describes the analysis of protein cysteine redox status. Redox status is crucial in regulating protein activity, stability, and redox signaling cascades. It is determined by conjugation with 1.24 kDa MM(PEG)24 molecule to each reduced cysteine followed by western blot analysis. This protocol is easy to follow, and most of the reagents and instruments required are of common use in any lab. This protocol can be successfully applied to other biological sources.

Phosphorus (P) limitation in the majority of world soils is a major constraint for plant growth and crop productivity. RNA sequencing was used to discover novel P-responsive gene transcripts (PRGT) in leaves and roots of Arabidopsis. Hisat StringTie and Cufflinks TopHat transcript assembler were used to analyze reads and identify 1,074 PRGTs with a >5-fold altered abundance during P-limitation. Interestingly, 60% of these transcripts were not previously reported. Among the novel PRGT, 106 were from unannotated genes, and some were among the most P-responsive, including At2g36727 which encodes a novel microRNA. Annotated novel PRGTs encode for transcription factors, microRNAs, small signaling peptides, long non-coding RNAs, defense-related proteins, and transporters, along with proteins involved in many biological processes. We identified several genes that undergo alternative splicing during P-limitation, including a novel miR399 resistant splice variant of PHOSPHATE2 ( PHO2 . 2 ). Several novel P-responsive genes were regulated by PHOSPHATE STARVATION RESPONSE1 ( PHR1 ), PHR1-LIKE 1 ( PHL1 ) and PHO2 . We discovered that P-limited plants show increased resistance to pathogens and drought stress mediated by PHR1-PHL1. Identification of novel P-responsive transcripts and the discovery of the influence of P-limitation on biotic and abiotic stress adds a significant component to our understanding of plant P-signaling. Highlight: Phosphorus limitation elicits the expression of several novel genes including many previously unannotated genes, noncoding RNAs, small peptides and alternatively spliced RNAs, and leads to enhanced disease and drought tolerance.