Roles of single gene in plant hypoxia and pathogen responses

ABSTRACT

Hypoxia stress can be caused by submergence or pathogen infection. These two stresses often occur sequentially or at the same time in nature. Therefore, plants have evolved economical and efficient strategies to deal with them, such as “single-gene multi-functions”, that is, one gene could play roles in hypoxia or pathogen responses at the corresponding stress. This review mainly introduces the ERF-VII (ethylene response factor VII) and WRKYs (WRKY transcription factors) that can play roles in these two stresses. Meanwhile, the relationship between hypoxia and pathology has certain similarities in animals and plants, so we can learn from their related studies and develop new ideas for disease therapy and breeding.

Introduction

Hypoxia is considered as a double-edged sword for it is not only an abiotic stress but also a development signal.^{1, 2} Hypoxia stress often is caused by waterlogging or submergence and will affect the growth and yields of plants. Flooding is a compound stress, mainly including hypoxia and pathogen stress. Multiple signaling pathways and transcriptional regulations are triggered, and ERF-VII transcription factors (including five members: HRE1, HRE2, RAP2.2, RAP2.3, RAP2.12)³ take a highlight position for its core role in oxygen sensing and signaling.^4,5 ERFVIIs have been linked also with other abiotic stresses too.⁶ Our previous research “WRKY33 interacts with WRKY12 protein to up-regulate RAP2.2 during submergence-induced hypoxia response in Arabidopsis thaliana” demonstrated the interplay between RAP2.2 and the two WRKYs attributed to adaptation to submergence-induced hypoxia stress in Arabidopsis.⁷ Interestingly, these genes are all known for their positive roles in plant pathogens response,^8–10 and flooding will increase plants’ susceptibility to pathogens,¹¹ then defense and hypoxia are likely correlated with the WRKY network. Corresponding to this, Botrytis cinerea infection generates local hypoxia in the leaf,¹² in the case of tumorigenic root tissues or gall-forming pathogens Plasmodiophora brassicae and Agrobacterium tumefaciens, plant responds to the infection by activating various hypoxia-related genes.^13,14 That inspires us to think the relationship between hypoxia and pathogen responses. Another review published last year discussed the impact of hypoxia on cellular physiology in fungal pathogens,¹⁵ while did not introduce detailed functions of the related genes. Plants incline to choose the most economical way that one-to-many or single-multifunctional-gene to regulate the development and stress responses, or the activation of innate immunity could have coevolved with submergence response,¹⁶ so what roles of the ERF-VII play in defense response? Here, we summarize these multifunctional genes response both in hypoxia and pathogen response (Table 1) and their potential mechanisms.

Table 1.

The biological roles of some genes related to both hypoxia and pathogen response in Arabidopsis, rice and barley.

Genes	Loc	Hypoxia response	Pathogen tolerance	Reference
AtWRKY33	AT2G38470	Positively	Positive against necrotrophic pathogens; negative against P. syringae.	7,9,17
AtWRKY12	AT2G44745	Positively	Suppressed expression during Pcc infiltration	7,8
AtWRKY22	AT4G01250	Positively	Positive against P. syringae	11
OsWRKY62	Os09g25070	Positively	Positive against M. oryzae and Xoo	18
AtRAP2.2	AT3G14230	Positively	Positive against B. cinerea	8,19
AtRAP2.3	AT3G16770	Positively	Positive against P. carotovorum	20,21
AtHRE1	AT1G72360	Positively	Positive against Pst DC3000	6,22
AtHRE2	AT2G47520	Positively	Positive against Pst DC3000	6,12,23
AtPRT6 HvPRT6	AT5G02310	Negatively Negatively	Negative against Pst DC3000 and P. brassicae Negative against Ps. japonica and Blumeria graminis f. sp. hordei	6,13 6
AtNTAQ1	AT2G41760	Negatively	Negative against Pst DC3000	6
AtATE1/2	AT5G05700 AT3G11240	Negatively	Negative against P. brassicae	4,13
AtMYC2	AT1G32640	Positively	Negative against B. cinerea and P. cucumerina	24,25
AtMPK3/6	AT3G45640 AT2G43790	Induced expression	Positive against Pto AvrRpt2	26,27
AtHUP26	AT3G10020	Induced expression	Positive against Pst DC3000	28,29
AtPCO1	AT5G15120	Positively	Induced expression	12,30
AtADH1	AT1G77120	Positively	Negative against P. brassicae	13,31
AtPDC1/2	AT4G33070 AT5G54960	Positively	Negative against P. brassicae	13
AtR8 Long Non-Coding RNA		Suppressed expression	Negative against P. syringae	32

RAP2.2 positively participates in necrotrophic fungus B. cinerea response dependent on the ethylene (ET) signaling pathway in plant,⁸ phenotypic analyses revealed that RAP2.2-overexpression and rap2.2 plants showed less and more severely damaged after B. cinerea infection, respectively. While the overexpression of RAP2.12 did not enhance tolerance to the fungus.¹² Our research⁷ proved that the WRKY33 and WRKY12 protein can up-regulate RAP2.2 by directly bind its promoter during submergence, moreover, RAP2.2 feedback up-regulate WRKY33 with the similar regulation way. This indicates that the pathogen resistance of RAP2.2 probably due to its interaction with WRKY33, which plays a key role in disease resistance and can regulate ethylene synthesis,^33–35 rather than be a partner of Med25.³⁶ RAP2.3 was demonstrated to interact with ORA59²¹, one of the best characterized ERF TFs involved in B. cinerea resistance, the lines overexpressing of RAP2.3 also showed resistance against pathogen while rap2.3 showed a hyposensitive phenotype.

The pentuple mutant erfVII lacks the entire Group VII ERF (HRE1 and HRE2 were included) showed increased susceptibility to sprayed Pst DC3000. HRE2 induction was also observed in Arabidopsis after infection with B. cinereal but not after treated with flg22¹², suggesting that the induction is due to local hypoxia caused by O₂ consumption by the living fungus. In our study,⁷ the expression of WRKY33 in hre1hre2 double mutant was much lower than in WT after submergence treatment, indicating the possible role for their regulation on WRKY33. By analyzing the resistance mechanism of the other ERF-VII members, the directly interplay with other defense key genes is required research in the future.

N-terminal cysteine oxidation of ERF-VII is enzymatically controlled by the PLANT CYSTEINE OXIDASES (PCOs) as part of the PRT6 N-degron pathway,^4,30 the content of nitric oxide (NO) and O₂ are key elements for their degradation. In hypoxia, not only NO and O₂ changed in levels, but also some other gaseous compounds, such CO_2,³⁷ ethylene^38,39 and H₂S.⁴⁰ They act a conserved role in transducing hypoxia response in plants and animals, as well as in influencing plant immune responses.⁶ Genes belonging to the N-degron pathway may thus provide a target for breeders to produce crops that increase disease resistance.⁶ For example, it is a common feature of pathogen induced tumor that activates of hypoxia responsive genes. In Homo sapiens, tumor hypoxia can be regulated by HIF (Hypoxia-Inducible Factor) pathway, which is similar with ERF-VII/N-degron pathway,^41,42 the hypoxia therapy can target on HIF response by small-molecule inhibitors or medications¹. Besides the oxygen-sensing N-degron pathway, the similarity of cellular hypoxia adaptations between the plants and animals is also remarkable,^1,20 such as MAPK pathway,⁴³ lipids,^44,45 non-coding RNAs,^32,46,47 and gaseous intracellular signal transducer.^22,48 The H₂S modulated PHD2/HIF-1α/MAPK signaling pathway to regulate tumor angiogenesis and cell injury;^49,50 plant pathogenic bacteria Xylella fastidiosa and A.tumefaciens utilize biofilm growth-associated repressor (BigR) to control H₂S detoxification under hypoxia.⁵¹ H₂S can enhance NO-induced tolerance of hypoxia in maize.⁴⁰ H₂S biosynthetic enzyme (DES1) mediates a crosstalk between H₂S, NO and ABA to control the stomatal closure.⁵² The erfVII mutants failed to close stomata in response to hemibiotroph pathogen Pst DC3000, but prt6-1 and ntaq1-3 did.⁶ It also shows the factors that can regulate or interact with key resistance-related genes are potentially involved in both defense and hypoxia responses, like HUP26 (HYPOXIA RESPONSE UNKNOWN PROTEIN 26),²⁸ HUP29(PCO1),¹² AtR8 Long Non-Coding RNA.³²

Another study⁵³ we recently reported revealed that the on-and-off module SR1-WRKY33-RAP2.2 is connected to the N-degron pathway to regulate submergence and reoxygenation responses in Arabidopsis. WRKY33 is phosphorylated by PA-MPK3/MPK6, WRKY33-P is simultaneously degraded by ubiquitin E3 ligase SR1 (SUBMERGENCE RESISTANT1) to regulate submergence response in Arabidopsis. However, there are still many questions needs to explore, such as how does SR1 sense oxygen levels to initialize the ubiquitination of WRKY33, and how is it removed when submergence ends? Is SR1 another oxygen sensor? In plant defense response, MAPK signaling could regulate the establishment of SAR (systemic acquired resistance) by regulatory loop consisting of WRKY33 and others.²⁶ It added the connection and deep understanding between the two known oxygen signal pathways. And the on-and-off module is similarly with the “growth–defense trade-off” rule in plant for they both reflect the plant’s fast and appropriate responses to the changing environment. OsWRKY62 also was reported to play roles in the trade-off between defense and hypoxia responses by regulating different factors at a given time point.¹⁸

Future challenges

A single TF regulates different target genes depending on different environmental conditions, this is optimized for cultivation of crops under given conditions. Future research should aim to unravel more details about the crossing points genes between the signal pathways, especially the genes interaction between N-degron pathway and key resistance-related factors, and the research results in both plants and animals can give a hint to each other in disease resistance or breeding for compound stresses, then help to provide effective survival strategies for all of us.

Funding Statement

This work was supported by the The National Natural Science Foundation of China [31870244].

Abbreviations

P. syringae

Pseudomonas syringae

Pcc

Pectobacterium carotovorum ssp. carotovorum

Pst DC3000

P. syringae pv tomato DC3000

MAPK

mitogen activated protein kinase

H₂S

Hydrogen Sulfide