Plant Stomata Function in Innate Immunity against Bacterial Invasion

Introduction

Plant bacteria can multiply under favourable conditions causing many serious diseases of crops which may have major economic impacts. Bacteria live in the phyllosphere of the plant, which is the niche for their colonization and survival. Bacteria live as epiphytes on the surface of the leaves. Pathogens must enter the leaves to initiate pathogenesis and access host nutrients.

Unlike fungi they cannot penetrate the leaf epidermis directly and need some natural openings or wounds to get in. Various natural openings in plants besides stomata include hydathodes in leaf margins, nectarthodes in flowers and lenticels in stems and roots.Stomata dominate in number amongst other natural openings in plant’s aerial part. These microscopic openings are made of specialized epidermal cells called guard cells. Opening and closing is regulated by change in turgor pressure in these guard cells. Also environmental cues like light, amount of CO2, drought stress and humitdity affect changes in stomatal aperture. The following picture shows what an open and a closed stomata look like.

Plant solely rely on their innate immune system to defend them against invading pathogens. Plant bacterial pathogens are revealed to their host by molecules called PAMPs i.e. Pathogen Assisted Molecular Patterns. These include flagellin, lipopolysaccharides and Elongation Factor EF-Tu. Plants have evolved specialized cell surface receptors to detect conserved features of PAMPs called Pattern Recognition Receptors (PRRs).

Bacteria have a twenty-two amino acid peptide derived from bacterial flagellin called flg22. This is detected in Arabidopsis by a PRR FLS2 (Flagellin Sensing 2), transmembrane receptor kinase. The FLS2 protein is expressed in epidermal cells and stomatal guard cells.

EF-Tu is the most abundant protein in a growing bacterial cell induces basal defences. It is a 43 kDa protein which has an 18 amino acid N-terminal peptide, elf18, is the key component. It’s a recognition occur by Receptor Like Kinase (RLK) named EFR (EF-Tu receptor)

Lipopolysaccharide is a major component of outer membrane Gram-negative bacteria. It is a glycolipid that has three different domains: lipid A, conserved inner core oligosaccharide , variable outer polysaccharide. Out of these lipid A acts as a PAMP.

TTSS : type three secretion system is responsible for Hypersensitive response. It results in localized tissue necrosis, production of phenolics and other anitmicrobial agent. Whether bacterial infection leads to plant diseases or to an Hypersensitive response is determined largely resistant gene (R) in the host and avr gene (avirulence gene) in previously resistant plants and induction of an R gene into a susceptible plant confers

resistance to pathogen with avr locus. Type III secretion system : a virulence factor is conserved in the four major genera of plant-pathogenic bacteria, Erwinia, Pseudomonas, Ralstonia, and Xanthomonas

Pseudomonas syringae pv tomato DC3000Coronatine

Phytotoxin Coronatine is produced by atleast five different pathovars of P.syringae. it is a non host specific polyketide toxin. It is required for full virulence of COR producing P.syringae.COR is structurally similar to family of plant compounds known as jasmonates. Studies show that COR simulates jasmonate responses in arabidopsis. Though it is one of the most well studied bacterial phytotoxins the precise mechanism by which it works is still unclear.

Authors first started by proving that bacteria and PAMPs trigger stomatal closure. They did experiments on both intact leaves and epidermal peels. They took Arabidopsis ecotype col-0 and inoculates with pstDC3000, thereby they observed number of open and closed stomata at different time intervals. As a negative control they also kept leaves in water. Besides to inoculated leaves’ epidermal peels with E.Coli, to see if this innate immunity can respond towards to different bacteria. They observed closure of stomata within 2 hours in leaves and within 1 hour in epidermal peels. However, E.Coli wasn’t able to reopen the stomata although it caused closing of stomata in the same way as PstDC3000.

In the next set of experiments the authors checked the dependence of closure of stomata on fls 2 receptor and salicylic acid. SA concentration increases at the site of wound infection which could possibly be another reason for innate immunity.

In the first set they checked the average width of stomatal aperture in a suitable MES buffer as control and flg22 and LPS as test conditions. As expected there was a decrease in width of the latter.Second test shows the width of stomatal aperture in wild type and fls2 mutant. Flg22 failed to close stomata in the mutant whereas LPS did.Also E.coli did induce closure of stomata in fls2 mutant. This suggested that guard cells perception of flg22 requires fls2 receptor but this is only one of the various other PAMPs on the bacterial surface.Finally for Sa]A dependence they checked wild type plants woth the SA deficient nahg transgenics and SA biosynthetic mutants eds16-2 and stomatal closure was compromised in both. Hence the authors concluded that stomatal closure is an integral part of SA regulated innate immune system

ABA signalling regulates stomatal closure and inhibition of stomatal opening. Therefore authors checked stomatal respinse to bacterial PAMPs in ost1 kinase mutant. Ost1 kinase is one of the downstream signal transduction component of ABA signalling.Neither flg22 nor LPS was able to induce NO production in guard cells. Here you can see NO production in Col-0 plants treated with MES buffer, flg22 peptide and LPS. There wasn’t any NO in control plant treated with MES buffer.Besides, they also testec L-NNA i.e. N-nitro –L- Arginine an inhibitor of Nitric oxide synthase (NOS) prevent stomatal closure in both PstDC3000 and E.coli that was induced by flg22 and LPS.

As observed in 1st set of experiments there was a reopening of stomata by PstDC3000. In contrast E.coli couldn’t induce such a response. This behaviour of associated with some virulence mechanisms that could counter Pamo induced stomatal closure. PstDC3000 is known to contaon 2 virulence factors : TTSS and COR

As seen in these sets of data cor mutant (strain DC3118) was not able to open stomata as the DC3000 and behaved in the same way as E.coli. However, hrcC i.e. TTSS mutant wasn’t affected in its ability to reopen stomata. Therefore, virulence factor that can be associated with suppressing stomatal defence is coronatine. This was supported by observing stomatal responses in these cases. Clearly COR was responsible for stomatal reopening.

To answer a critical question i.e. PAMP induced stomatal closure result in effective restriction of bacterial entry through the epidermis; authors conducted a certain set of experiments.

They placed GFP labelled DC3000 and COR mutant DC3118 bacteria underneath an epidermal peel and incubated for 3 hrs. they observed PstDC3000 on upper surface of the

peel and formed characteristic cultures. On the other hand, the ones treated with cor mutants were devoid of any bacterial clusters.

In another set experiments suspensions of PstDC3000 and DC3118 were vacuum infiltrated into Col-0 plants. After three days bacterial growth was observed which was greater by almost a factor of 103

For eds16-2 and nahG transgenics growth was very high for virulent strain DC3000 and was relatively lower for cor mutant DC3118.

Similarly for aba 3-1 and ost1 mutant cor mutant multiplied to levels similar to that reached by wild type bacterium at day 3 and caused disease symptoms. These results suggest that suppression of stomatal defence is the primary function of COR in local leaves

Authors suggest a model for bacteria and PAMP induced stomatal closure in Arabidopsis guard cells. They propose PAMPs displayed by bacteria are perceived by specific receptors in stomatal guard cells. PAMP recognition by PRR links to ABA regulated stomatal closure.The virulence factor COR secreted by PstDC3000 interferes with stomatal closure. Action of Cor is dependent on coi1, a subunit of E3 ubiquitin ligase also involved in signalling of the plant defence hormone jasmonic acid.

SUMMARY

To sum up the research done by authors: plants have ability to defend themselves against pathogenic bacteria by closing ports of entry for bacteria i.e. stomata.Stomatal closure could be linked to SA mediated innate immunity system of plants.Plants pathogens have evolved specific virulence factors to trick guard cells to suppress stomatal defence and open entry channels for themselves to penetrate through.

QUESTIONS PROMPTED

Do other plants have such stomatal defence? Authors investigated response of tomato stomata to PstDC3000 and LPS. They were similar to Arabidopsis.

Only a few pathovars produce COR if stomatal defence widespread, some vir factors or lifestyles to overcome (in bacteria)Pst pathovar tabaci doesn’t produce COR but was able to induce stomatal closure initially and reopen stomata same as PstDC3000. therefore stomata closure may be a common plant defence response initiated by perception of PAMPs and COR is one of the several vir factor used by bacteria to overcome these defences

This is likely to open up avenues for future research.

Do environmental factors that affect stomatal opening influence effectiveness of stomata based innate immunity. This may lead to new understanding of outbreaks of bacterial diseases in crop plantsSeveral outbreaks associated with increases rain and increases humidity. In these conditions plants stomatal defence might be partially compromised due to high priority of stomatal response to other condition

What is the order of prioritization of events that lead to stomatal closure? Again PAMP perception acts through ABA signalling, its not clear whether perception by PAMP increases ABA biosynthesis or basal level of ABA is required by PAMP.

COR has shown to promote opening of in dark closed stomata : entry of bacteria at night when most stomata are closed. COR didn’t block NO synthesis. COR acts downstream or independent of NO synthesis. Therefore precise mode of action of COR can lead to better understanding of defence mechanism.