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CHAPTER NINE

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Evidence for Widespread Microbivory of Endophytic Bacteria in Roots of Vascular Plants Through Oxidative Degradation in Root Cell Periplasmic Spaces

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James F. White Jr.1, Mónica S. Torres1, Satish Kumar Verma2, Matthew T. Elmore1, Kurt P. Kowalski3, Kathryn L. Kingsley1 1 Department of Plant Biology, Rutgers University, New Brunswick, New Jersey, USA 2 Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, UP, India

3 U.S. Geological Survey, Great Lakes Science Center, 1451 Green Road, Ann Arbor, MI, USA

9.1. INTRODUCTION

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There is grow ing ev i dence that all plants are in hab ited by a plethora of mi crobes (Stone et al., 2000; Arnold and Lutzoni, 2007; Rosenblueth and MartínezRomero, 2006; Magnani et al., 2010; Compant et al., 2010; JohnstonMonje and Raizada, 2011). These com po nents of the plant mi cro biome are both bac te r ial and fun gal and may ex ist on plant sur faces and in te ri ors. How mi crobes in ter act with one an other, and with host plants, is cur rently not well un der stood, al though we do have frag men tary knowl edge. For ex am ple, re search on in di vid ual com po nents of the mi cro biome in di cates that the mi cro biome in hab i tants may en hance a host plant’s re sis tance to bi otic and abi otic stresses (Kloepper, 1993; Redman et al., 2002; Clay et al., 2005; Waller et al., 2005; Clarke et al., 2006; Weber et al., 2007; Kuldau and Bacon, 2008; Rodriguez et al., 2009; ÁlvarezLoayza et al., 2011; Bacon and Hinton, 2011; Hamilton et al., 2012; Torres et al., 2012; Doty, 2017). These stud ies clearly in di cate that the mi cro biome may pos sess some de fen sive prop er ties that ben e fit plant hosts. There are also in di ca tions that plant

PGPR Amelioration in Sustainable Agriculture ISBN 9780128158791 https://doi.org/10. 1016/ B9780128158791. 000094


© 2019.

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James F. White et al.

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mi cro bio mes pos sess nu tri tional prop er ties (Hurek et al., 1994; Döbereiner, 1992; Döbereiner et al., 1994; James et al., 1994; Puente and Bashan, 1994; Glick, 1995; James and Olivares, 1998; James, 2000; ReinholdHurek and Hurek, 2011). One such ex am ple is the phe nom e non of “as so cia tive ni tro gen fix a tion” where en do phytic di a zotrophic bac te ria in the mi cro biome fix ni tro gen and stim u late plant growth. Some sci en tists posit that this phe nom e non is re spon si ble for ef fi cient plant growth in crops such as sug ar cane, rice, wheat, and corn (James, 2000; Urguiaga et al., 1992; Taulé et al., 2012). How ever, most re search on plant mi cro biome nu tri tional ef fects on plant growth and de vel op ment to date has in volved ex per i ments con ducted on in di vid ual bac te ria that are ap plied to plants. Con se quently, we have lim ited in for ma tion re gard ing other mi cro biome in hab i tants. Pre cisely how host plants ob tain nu tri ents from the mi crobes that col o nize them has long been an unan swered ques tion (James, 2000). PaungfooLonhienne et al. (2010a, b) pro vided the first ev i dence of a mech a nism for the trans fer of nu tri ents from mi cro biome mi crobes to plants by demon strat ing that tomato plants and Ara bidop sis thaliana are ca pa ble of mi cro bivory through en do cy to sis and degra da tion of mi crobes within root cells. Mi cro bivory is gen er ally known to oc cur among het erotrophic pro to zoans and sim ple an i mals where the eu kary otic con sumer en gulfs and de grades bac te ria as a nu tri ent source (Mikola, 1998). More re cently, White et al. (2012) demon strated the ly sis of di a zotrophic bac te ria on sur faces of grass root hairs and root epi der mal tis sues. The process of ly sis in volved, at least in part, se cre tion of re ac tive oxy gen onto bac te ria to de grade/ ox i dize bac te ria, and for that rea son the process was termed “ox ida tive ni tro gen scav eng ing.” Col lec tively these stud ies sug gest that at least some plants have the ca pa bil ity to ac quire nu tri ents through the ly sis of mi crobes in the mi cro biome. More over, wide spread mi cro bivory in plants for nu tri ent ac qui si tion from mi crobes could have far reach ing con se quences. To be spe cific, un der stand ing this mech a nism could lead to the de vel op ment of new strate gies for plant cul ti va tion that use mi crobes as nu tri tional sup ple ments in stead of in or ganic fer til iz ers (Kraiser et al., 2011; PaungfooLonhienne et al., 2012).


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Evidence for Widespread Microbivory of bacteria

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9.2. SEEDLING SURVEY, SEED TRANSMISSION, AND BACTERIAL DISTRIBUTION IN SEEDLING TISSUES

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Bac te ria are seed trans mit ted, al though it is dif fi cult to de ter mine pre cisely where bac te ria are vec tored in seeds. There are two op tions: 1. Bac te ria may be em bed ded on/ in the seed sur face lay ers, per haps in re sis tant biofilms; or 2. Bac te ria may oc cur within seeds, per haps in the em bryo it self (Frank et al., 2017; Rodríguez et al., 2017). In the grasses rig or ous dis in fec tion ap peared to dras ti cally re duce bac te ria in seedling root tis sues (White et al., 2015; White et al., un pub lished). This sug gests that most of the bac te ria in these plants are vec tored on the sur face of seeds (White et al., 2012). It is pos si ble that seedlings of all plant species may also re cruit bac te ria from the en vi ron ment. Seedlings grow ing in soils may ac quire di verse re cruited en vi ron men tal bac te ria (Frank et al., 2017; Rodríguez et al., 2017). Fu ture stud ies will be needed to re solve ques tions of the ecolo gies of the seedling bac te ria. We con ducted a sur vey of seedlings of 36 species of plants by col lect ing seeds from nu mer ous sources, wash ing them with con tin u ous ag i ta tion in three changes of ster ile wa ter (5 mintues each change) to re move soil and de bris, ger mi nat ing them on agarose and stain ing them in 2.5 mM di aminoben zi dine tetra chlo ride (DAB; SigmaAldrich, USA) by flood ing plates with the stain for 10–12 h. DABstained roots were counter stained with ani line blue and ob served un der the light mi cro scope (White et al., 2014; White et al., 2017). DAB en ables vi su al iza tion of re ac tive oxy gen pro duced around in tra cel lu lar bac te ria (White et al., 2014), show ing both pres ence of bac te ria and ac tion of re ac tive oxy gen on them. Ani line blue stains pro teins in bac te r ial cy to plasm and shows bluestained bac te r ial con tents in bac te ria that are not fully ox i dized; swollen bac te ria with out in ter nal blue stain ing in di cates fully ox i dized bac te ria. For Poly podium poly po di oides young plants were col lected from nat ural pop u la tions, stained, and ex am ined mi cro scop i cally. In our sur vey of seedlings, bac te ria were mostly found in root tis sues —but were also some times ob served in shoot tis sues (Table 9.1). In roots, the bac te ria were pre sent fre quently in parenchyma and root hair cells lo cated in the periplas mic space be neath the cell walls where they were seen to lyse [Fig. 9.1(C–F), Fig. 9.2(A–D), Fig. 9.3(B), Fig.


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TABLE 9.1 Sur vey of plant species for ev i dence of ox i da tion/ degra da tion of bac te ria in seedling root cells Family

Origin

Bacteria isolated

Agave chrysantha

Agavaceae

Sonoran desert, AZ

Unidentified

Agave palmeri

Agavaceae

Sonoran desert, AZ

Klebsiella sp. (White et al., 2014)

Agave schottii

Agavaceae

Sonoran desert, AZ

Unidentified

Amaranthus viridis

Amaranthaceae Commercial Unidentified source, USA

Apium graveolens

Apiaceae

Brassica napus

Brassicaceae

Commercial Unidentified source, USA

Celastrus orbiculatus

Celastraceae

East Brunswick, New Jersey

Unidentified

Coriandrum sativum

Apiaceae

Commercial source, Mexico

Unidentified

Cucurbita pepo

Cucurbitaceae


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Cells showing bacterial degradation

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Species


Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap Root hairs, Root epidermis Root hairs, Root epidermis Root hairs, Root epidermis Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap

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TABLE 9.1 (Continued) Family

Origin

Bacteria isolated

Citrullus colocynthis

Cucurbitaceae

Commercial source, Nigeria

Unidentified

Cereus repandus

Cactaceae

Cynodon dactylon

Poaceae

Dahlia sp.

Asteraceae

Fallopia japonica

Polygonaceae

Fimbristylis cymosa

Cyperaceae

Festuca arundinacea

Poaceae

Hedera helix

Araliaceae

Leersia oryzoides

Poaceae

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Root hairs, Root epidermis, Root cap Bonaire, Achromobacter Root hairs, Dutch xylosoxidans Root Antilles (White et al., epidermis, 2014) Root cap Commercial Unidentified Root hairs, source, USA Root epidermis, Root cap Commercial Bacillus sp. (Li et Root hairs, source, USA al., 2015) Root epidermis South River, Bosea thiooxidans Root hairs, New Jersey, (White et al., Root USA 2017) epidermis Bonaire, Unknown Root hairs, Dutch Root Antilles epidermis, Root cap Commercial, Pantoea Root hairs, USA agglomerans Root White et al. (2012) epidermis New Jersey, Bacillus Root hairs, USA amyloliquefaciens Root (Soares et al., epidermis 2015) New Jersey, Pantoea spp., Root hairs, USA Pseudomonas sp. Root (Verma et al., epidermis, 2017b) Root cap Commercial Bacillus sp. Root hairs, source, USA Root epidermis

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Lolium perenne

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Species


Poaceae


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Origin

Bacteria isolated

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Poaceae

Poa annua

Poaceae

East Brunswick, New Jersey, USA Penn State Cultivar Selection, From David Huff Commercial source, USA

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Panicum virgatum

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Lonicera Caprifoliaceae New Jersey, Unknown japonica USA Lycopersicum Solanaceae Commercial Acinetobacter sp., esculentum source, USA Micrococcus luteus White, (Unpublished) Moringa Moringaceae Commercial Citrobacter sp., oleifera source, USA Bacillus sp., Klebsiella sp. (White, Unpublished) Oryza sativa Poaceae Commercial Verma et al. source, USA (2017a)

Phaseolus acutifolius

Fabaceae

Phragmites australis

Poaceae

New Jersey, USA

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Polypodium Polypodiaceae Panama polypodioides

Burkholderia sp. (White et al., 2014) Unidentified

Root hairs

Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap Root hairs, Root epidermis, Root cap Root hairs, Root epidermis Root hairs, Root epidermis, Root cap

Unidentified

Root hairs, Root epidermis, Root cap Pseudomonas Root hairs, spp., Pantoea sp. Root White et al. (2017) epidermis, Root cap Unidentified Root hairs


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Origin

Rhus radicans

Anacardiaceae Middlesex Co., New Jersey, USA

Polygonaceae

Taraxacum officinale

Asteraceae

Triticum aestivum

Poaceae

Typha latifolia

Typhaceae

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Rumex crispus

Sphingomonas sp. Root hairs, (White et al., Root 2014) epidermis, Root cap Bonaire, Unidentified Root hairs, Dutch Root Antilles epidermis New Jersey, Unidentified Root hairs, USA Root epidermis, Root cap South River, Unidentified Root hairs, New Jersey, Root USA epidermis, Root cap Commercial Unidentified Root hairs, source, USA Root epidermis, Root cap New Unidentified Root hairs, Brunswick, Root New Jersey, epidermis, USA Root cap New Jersey Unidentified Root hairs, Root epidermis, Root cap Sonoran Klebsiella sp. Root hairs, desert, USA White et al. (2014) Root epidermis, Root cap

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Ritterocereus Cactaceae griseus

Bacteria isolated

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Species


Ericaceae

Yucca schottii

Agavaceae

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Vaccinium oxycoccos

9.4(D,E), Fig. 9.5(B–E)]. Thomas and Reddy (2013) ob served col o niza tion of periplas mic spaces of sev eral lines of ba nanas (See also Thomas and Soly, 2009). In root tis sues, nonl ysed bac te ria were of ten lo cated closer to the root tip meris tem [Fig. 9.1(a,b)] with swelling and ly sis of bac te ria more pro nounced in cells as they dif fer en ti ated [Fig. 9.1(c–f)].


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Figure 9.1 (A–F) Cat tail (Ty pha an gus ti fo lia) seedling ger mi nated on agarose. (A) Root tip show ing pink cloud of bac te ria sur round ing the meris tem in the zone of in tra cel lu lar col o niza tion (bar = 1 mm). (B) Epi der mal cell near the root tip meris tem show ing blue stained bac te ria (ar rows) in the periplas mic space of the epi der mal cell. In tra cel lu lar bac te ria are ev i dent as small blue specks on the plasma mem brane of the cell (bar = 10 µm). (C–E) Root axis show ing epi der mal parenchyma cells con tain ing brown clus ters of ox i diz ing bac te ria (ar rows) in the periplas mic space of cells. Root was stained with DAB to vi su al ize re ac tive oxy gen around ox i diz ing bac te ria (bar = 10 µm). (F) Root hair show ing in ter nal clus ter of ox i diz ing bac te ria (ar row) in the periplas mic space; the tis sue was stained with DAB to show H2O2 (brown).


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Figure 9.2 (A–D) Roots be long ing to species of fam ily Cu cur bitaceae show ing in ter nal ox i diz ing bac te ria. (A and B) Root cor tex cells show ing ox i diz ing bac te ria (ar rows) in the periplas mic spaces of roots of egusi melon (Cit rul lus colo cyn this) stained with DAB (bar = 10 µm). (C) Root cor tex cells show ing ox i diz ing bac te ria (ar rows) in the periplas mic spaces of roots of acorn squash (Cu cur bita pepo) (bar = 10 µm) stained with DAB. (D) Sloughedoff root cap cell of acorn squash seedling stained with DAB show ing ox i diz ing bac te ria (ar row) in the periplas mic space (bar = 10 µm).

This pat tern of dis tri b u tion of bac te ria may be an in di ca tion that bac te ria pro lif er ate around the root tip mer sitem of seedlings [see Fig. 9.1(a)] and col o nize the meris tem atic cells [Fig. 9.1(b)] where cell walls are rel a tively un de vel oped. Bac te ria that pro lif er ate in root and shoottip meris


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Figure 9.3 (A–F) Eng lish Ivy (Hed era he lix) show ing root hairs with in tra cel lu lar bac te ria. (A) Root of Eng lish ivy prop a gated in the lab o ra tory from cut tings then stained with DAB to show high re ac tive oxy gen ac tiv ity (brown col oration) in the lat eral roots (ar rows) that bear nu mer ous root hairs (bar = 1 cm). (B) Root hair ini tial (ar row) show ing high re ac tive oxy gen ac tiv ity (brown col oration) and in ter nal ox i diz ing spher i cal bac te ria (bar = 10 µm). (C) Root hair ini tials (ar row) show ing nu mer ous in ter nal bac te ria (bar = 10 µm). (D) Root hair ini tial show ing bac te r ial Lforms (ar row) ex it ing hair at the tip (bar = 10 µm). (E) Root hair ini tial show ing nu mer ous in ter nal bac te ria (black ar row) clus tered in the periplas mic space at the hair tip (bar = 10 µm). Exit pores (white ar rows) are vis i ble in wall at the hair tip. (F) Root hair show ing bac te ria (ar row) be neath the wall at the hair tip (bar = 10 µm).


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Figure 9.4 (A–F) Poi son Ivy (Rhus rad i cans). (A) Seedling of Rhus rad i cans grow ing on agarose show ing char ac ter is tic red roots (ar row) (bar = 1 cm). (B) Roots grow ing in agarose show ing nu mer ous root hairs (ar row) (bar = 1 mm). (C) Root hair (ar row) show ing gran u lar ap pear ance in ter nally due to in ter nal bac te ria (bar = 10 µm). (D). Root hair stained with DAB show ing bac te ria (ar row) in a vesi cle sur rounded by red to brown ring of re ac tive oxy gen (bar = 10 µm). (E) Root axis parenchyma cell show ing ox i diz ing bac te r ial Lforms (ar row) in the periplas mic space (bar = 10 µm). (F) Sloughedoff root cap cell show ing bac te ria (ar rows) in ter nally (bar = 10 µm).


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Figure 9.5 (A–E) Corn (Zea mays) root cells stained with DAB and ani line blue to show in ter nal bac te ria. (A) Sloughedoff root cap cells of mod ern white corn seedling show ing ab sence of in ter nal bac te ria in the periplas mic space of cells (bar = 10 µm). (B) Root cap cells from trop i cal corn seedling show ing bac te ria (ar rows) in vesi cles with a ring of re ac tive oxy gen (red dish ring) sur round ing bac te r ial cells (bar = 10 µm). (C–E) Root epi der mal parenchyma cells from trop i cal corn seedling stained with DAB show ing ox i diz ing bac te ria (ar rows) in cells (bar = 15 µm).


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tems are dis trib uted in all parts of the plant; when plants pro duce fruits they may be col o nized by the en dosym bi otic bac te ria.

9.3. EVIDENCE FOR MICROBIVORY IN DIVERSE VASCULAR PLANT FAMILIES

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In our sur vey, we found ev i dence of in tra cel lu lar bac te ria in all species ex am ined [Table 9.1; Fig. 9.1(B–F), Fig. 9.2(A–D), Fig. 9.3(B), Fig. 9.4(D, E), Fig. 9.5(B–E)], in clud ing some 36 species of plants dis trib uted in 20 fam i lies of vas cu lar plants. Ferns and seed plants were found to in ter nal ize and ox i dize bac te ria in root cells. In all species, bac te ria ap peared to in ter nal ize in root cells at root tip meris tem as walled cells, only to be come wallless Lforms once in side the periplas mic space be tween plant cell wall and plasma mem brane [Fig. 9.2(A–D)]. Even aer ial roots of vines, in clud ing Eng lish Ivy (Hed era he lix) and Poi son Ivy (Rhus rad i cans), were seen to in ter nal ize and ox ida tivelyde grade bac te ria within root cells [Fig. 9.3(A–F), Fig. 9.4(A–F)]. An ex cep tion was white corn (Zea mays), a mod ern corn hy brid, where ex am i na tion of seedlings did not re veal abun dant bac te r ial in ter nal iza tion in root cells [Fig. 9.4(f)]. How ever, trop i cal corn (Zea mays) and In dian corn (Zea mays), both less in ten sively se lected than mod ern hy brids, were found to con tain abun dant bac te ria in seedling root cells [Fig. 9.5(B–E)]. It could be that the high de pen dence of mod ern hy brid corn va ri eties on in or ganic fer til iz ers may be the re sult of the loss of sym bi otic bac te ria that func tion carry nu tri ents into plants.

9.4. NUCLEAR COLONIZATION

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Bac te ria re sisted degra da tion longer when they en tered nu clei of plant cells. Lysed bac te ria were fre quently found in cell cy to plasm just out side the nu clear en ve lope [Fig. 9.5(c)] and in tact bac te ria within the nu clei. It is pos si ble that bac te ria sur vive longer within nu clei be cause the nu cleus is an area of the cell where re ac tive oxy gen lev els are low and ly sis gen er ally does not oc cur. Pos si bly, bac te ria may pro duce nu cle o mod ulins to con trol gene ex pres sion of the cell to re duce ox ida tive processes or al ter ac tiv i ties of the cell in a way that fa vors the en dosym bi otic bac te ria (Bierne and Cossart, 2012). For ex am ple, nu cle o mod ulins are pro duced by Agrobac terium tume fa ciens, an other species of Pro


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teobac te ria. Nu clear col o niza tion also oc curs in pro to zoans where al pha Pro teobac te ria of the fam ily Holospo raceae are phago cy tized by the host pro to zoan and trans ported to the nu cleus where bac te ria mul ti ply. Nu clear col o niza tion by bac te ria was also de scribed in the sym bi otic sys tem in volv ing en donu clear betaPro teobac te ria and the di nofla ge late Gy ro dinium in s tria tum (Alverca et al., 2002). In this sys tem, di vid ing bac te ria were ob served in the nu cleus. In tranu clear bac te ria were re leased to the cy to plasm where they were of ten de graded (Alverca et al., 2002). These au thors also pro posed that nu clear col o niza tion was a strat egy to es cape di ges tion that oc curred in the cy to plasm of the alga. It is pos si ble that bac te ria in root cells may en ter nu clei as a way to es cape re ac tive oxy gen degra da tion that oc curs on host cell mem branes in the cy to plasm.

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9.5. BACTERIAL MOVEMENT IN PLANT CELLS

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Re search on the Holospo raceae sug gests that the bac te ria are able to move through the host cell’s cy to plasm to the nu cleus through use of actin fil a ments (Sabaneyeva et al., 2009). How ever, this mech a nism may not be uni ver sal for in tra cel lu lar bac te ria. In mak ing ob ser va tions on un stained liv ing seedling roots of the sedge Fim bristylis cy mosa and the grass Fes tuca arun d i nacea move ment of bac te ria was ob served within root hairs. Bac te ria lo cated in the periplas mic space ap peared to flow along the length of root hairs. The rate of flow was 8–11 µm/ sec ond for the sedge F. cy mosa. This move ment may have been due to cy clo sis within the root hairs. PaungfooLonhienne et al. (2010a, b) also re ported move ment of in tra cel lu lar mi crobes in root hairs of A. thaliana. Bac te r ial move ment was not ob served in stained seedlings since stains DAB and ani line blue with lac tophe nol gen er ally stopped cy clo sis in cells. It is likely that move ment within cells is the norm for the in tra cel lu lar bac te ria. This in tra cel lu lar move ment may per mit the bac te ria to spread to all parts of the host cell. Con stant move ment of in tra cel lu lar bac te ria may also re duce the ef fects of re ac tive oxy gen and lysing en zymes on bac te ria.


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9.6. BACTERIAL COLONIZATION OF SEEDLING ROOTS OF PANICUM VIRGATUM

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Ex per i ments were con ducted to eval u ate col o niza tion of seedling roots us ing a strain of Burk holde ria glad i oli iso lated from ger mi nat ing seedlings of Pan icum vir ga tum (White et al., 2014). Through sur face dis in fec tion seeds free of, or with re duced lev els, of bac te ria were ob tained with which we con ducted seedling in fec tion ex per i ments. On in oc u la tion of seedlings with bac te r ial sus pen sions col o niza tion of seedling roots was ob served. This was ac com pa nied by a shape change in the struc ture of bac te ria from rod to sphereshaped (White et al., 2014). Paungfoo Lonhienne et al. (2010a, b) in a study of Es cherichia coli en try into Ara bidop sis seedlings found that en try into cells was ac com pa nied by up reg u la tion of the plant cell wallre lated en zymes, in clud ing ex pansins, cel lu lases, pecti nases, xy loglu can en do trans g ly cosi dases, and cel lu lose syn thases. In volve ment of host en zymes in the en try of bac te ria into cells sug gests that plant cells are en gag ing in phago cy to sis to ac quire bac te ria. How ever, it is also pos si ble, at least in some cases, that the bac te ria them selves pro duce cell wall loos en ing and de grad ing en zymes to col o nize the in te rior of plant cells. Other sym bi otic bac te ria are thought to en ter plant cells us ing their own cell wall de grad ing en zymes. Kovtunovych et al. (1999) demon strated that the ca pac ity of Kleb siella oxy toca to en do phyt i cally col o nize wheat plants cor re lated with its abil ity to pro duce pecti nases. Compant et al. (2005) demon strated that Burk holde ria sp. re quired use of cell wall de grad ing en zymes en doglu conase and poly galac tur onase to in ter nally col o nize grapes.

9.7. CHANGE IN BACTERIAL SHAPE

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The change of cell shape from rodlike to spher i cal for Burk holde ria cells col o niz ing seedling roots of P. vir ga tum is likely the re sult of in ter ac tion be tween the plant and bac terium (Beran et al., 2006). Shape trans for ma tions also oc cur in bac te ria that in tra cel lu larly col o nize an i mals (Beran et al., 2006). The spher i cal, of ten in tra cel lu lar, forms are re ferred to as Lforms or cell wall de fi cient forms (Beran et al., 2006). L forms of bac te ria are found in healthy in testi nal tracts of an i mals and have been im pli cated as causal agents of hu man dis eases such as Crohn’s


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dis ease, ul cer a tive col i tis, Sar coido sis, pul monary tu ber cu lo sis, Hodgkin’s dis ease, and sev eral other hu man dis eases (Wall et al., 1993; Beran et al., 2006). Bac te ria lose their cell walls due to loss of struc tural wall com po nents, re sult ing in spher i cal bac te r ial cells. It has also been sug gested that bac te ria form Lforms in or der to evade host de fenses (Beran et al., 2006). In plants, ar ti fi cially gen er ated Lforms of bac te ria have been shown to en ter seedling root tis sues and es tab lish in tra cel lu lar sym bi otic growth (Amijee et al., 1992; Daulagala and Allan, 2003). In sev eral plants es tab lish ment of en dosym bi otic Lforms of bac te ria re sulted in en hanced re sis tance to a range of plant pathogens, al though the mech a nism of en hanced re sis tance has not been clar i fied (Amijee et al., 1992). The ma jor ity of the in tra cel lu lar bac te ria we ob served in seedlings were spher i cal forms (Table 9.1). When bac te ria were iso lated from seedlings they were in vari ably rod forms in cul ture. Re ver sion to the walled cell shape is com mon when Lforms are iso lated onto nu tri ent me dia (Beran et al., 2006).

9.8. EVIDENCE FOR INCREASED NITROGEN ASSIMILATION BY BACTERIA IN PLANTA

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Re search on ni tro gen fix a tion by di a zotrophic bac te ria ap plied to plant tis sues has im pli cated roots rather than shoots as the lo ca tion where ni tro gen fix a tion (nif) genes are ex pressed in bac te ria, sug gest ing that bac te ria as so ci ated with roots are ac tive in ni tro gen fix a tion (Hurek et al., 1997; Rosenblueth and MartínezRomero, 2006). Soares et al. (2016) con ducted 15 N gastrack ing ex per i ments us ing plants of Phrag mites aus tralis with and with out ad di tion of en do phytic bac te ria and sim i larly found that in creased ni tro gen as sim i la tion into roots trans lated into in creased plant growth, while in creased as sim i la tion into leaves was not ac com pa nied by in creased growth. Whether the nat u rallyoc cur ring in tra cel lu lar bac te ria in seedling roots are ac tive in ni tro gen fix a tion is yet to be de ter mined. It seems ev i dent based on our ob ser va tions and those of oth ers that some of the nu tri ents ac quired by vas cu lar plants are ac quired through ly sis of bac te ria that be come in tra cel lu lar in roots (Beltrán García et al., 2014). Re search by PaungfooLonhienne et al. (2010a) is sug ges tive of a mech a nism in plants whereby they are adapted to ob tain nu tri ents from in tra cel lu lar bac te ria. These in ves ti ga tors found that in A.


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thaliana ex po sure of seedling roots to nu cleic acids turns on a path way that en ables plants to ob tain ni tro gen from pro teins (PaungfooLonhienne et al., 2010a). White et al. (2012) pre vi ously re ported that bac te ria are de graded on sur faces of grass roots through the ac tion of re ac tive oxy gen se creted from root tis sues onto bac te ria. One of the early ef fects of sur face ox i da tion of bac te ria was re lease of the nu cleic acids from bac te r ial cells. Nu cleic acids dif fused from bac te ria and ad hered to the root hair sur face sur round ing the lysing bac te ria (White et al., 2012). Nu cleic acid re lease from bac te ria could be a sig nal to plant cells to upreg u late pro teases and other cel lu lar pro tein degra da tion sys tems. Paungfoo Lonhienne et al. (2008) also demon strated that A. thaliana pos sessed pro te olytic en zymes that de grade pro tein on root sur faces and ac tively en gage in en do cy to sis of pro tein par ti cles. BeltránGarcía et al. (2014) la beled en do phytic bac te ria with 15 N iso tope then wa tered Agave tequi lana plants with a sus pen sion of vi able 15Nla beled bac te ria or heat killed 15Nla beled bac te ria and found that plants were able to as sim i late 15Nlabled ni tro gen from liv ing en do phyic bac te ria with a much greater ef fi ciency than from heatkilled bac te ria. PaungfooLonhienne et al. (2010a, b, 2013) de nom i nated the phe nom e non of in ter nal iza tion and degra da tion of mi crobes in cells of roots “rhi zophagy” or “root eat ing” to re flect the con cept that roots ac quire nu tri ents through a mi cro bivory process.

9.9. THE LYSIS PROCESS

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Con sis tent ob ser va tion of H2O2 ac tiv ity in and around de grad ing in tra cel lu lar bac te ria in seedling root tis sues sug gests that re ac tive oxy gen species (ROS) play an im por tant role in the ly sis process. In a study of bac te r ial ly sis on sur faces of seedling roots of F. arun d i naceae we pro posed that se creted ROS and plant pro teases func tioned to lyse bac te ria and their pro tein con tents to pro vide ni troge nous nu tri ents, a process we termed “ox ida tive ni tro gen scav eng ing” to em pha size ac qui si tion of ni tro gen from the process (White et al., 2012). The way that we en vi sion this process to func tion in in tra cel lu lar bac te ria, or bac te ria in the periplas mic spaces of cells, is that ROS, in clud ing su per ox ide pro duced by NADPH ox i dases on the plant cell plasma mem brane, or other host mem branes, is se creted into the vesi cles con tain ing bac te ria, or into the de pres sions in the plasma mem brane con tain ing bac te ria (White et al.,


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2018). The mem branes of plant cells are pro tected from ROS by sterols that pre vent pas sage of re ac tive oxy gen into the root cell it self (White et al., 2018). ROS de na tures bac te r ial walls and mem branes and en ters bac te r ial cells, dam ag ing pro teins and nu cleic acids and caus ing frag men ta tion of nu cleic acids that re sults in dif fu sion of nu cleic acid frag ments from the bac terium into the host cell’s cy to plasm (Kocha et al., 1997; Cabiscol et al., 2000). This is con sis tent with the ob ser va tions we made on de grad ing bac te ria on sur faces of, and within grass seedling root hairs (White et al., 2012). Nu cleic acid frag ments may act as sig nal mol e cules to stim u late the plant cell to se crete pro te olytic en zymes into vac uoles where pro tein dis ar tic u la tion is com pleted (PaungfooLonhienne et al., 2010a). Oligopep tides may then dif fuse into the cy to plasm where dis ar tic u la tion is com pleted. The ba sic mech a nism for pro tein degra da tion is likely to be sim i lar to the au tophagy process that is pre sent in all eu kary otes (Xiong et al., 2007). Au tophagy is the process whereby eu kary otes de grade their own pro teins that have been dam aged through ox i da tion. In plants, au tophagy gen er ally oc curs in vac uoles (Xiong et al., 2007). The au tophagy process seems con sis tent with our ob ser va tions in seedling roots where bac te ria in vesi cles were first ox i dized, re sult ing in en hanced pro tein stain ing us ing ani line blue stain, then a grad ual loss of ca pac ity to stain us ing ani line blue due to degra da tion of pro teins. In our sur vey (Table 9.1), we fre quently ob served the fu sion of smaller vesi cles to form larger vesi cles or vac uoles. Ul ti mately, these bac te r ial degra da tion vac uoles may be come part of the cen tral vac uole of the plant cell where au tophagy in more ma ture cells gen er ally oc curs.

9.10. MICROBIVORY AS A DEFENSE FROM PARASITISM BY ENDOPHYTIC BACTERIA

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It seems log i cal that mi cro bivory in vas cu lar plant roots is a de fense mech a nism against in tra cel lu lar in vad ing bac te ria. This idea is con sis tent with our cur rent un der stand ing of how eu kary otic cells em ploy re ac tive oxy gen as de fense against pathogens. In an i mals, re ac tive oxy gen is in volved in the killing and degra da tion of phago cytic leuko cytes (Robinson, 2008). In leuko cytes, the killing and degra da tion by hy dro gen per ox ide re sults from the for ma tion of more po tent ROS, in clud ing hy droxyl rad i cals, sin glet oxy gen, and ozone (Robinson, 2008). It is known that plants also se crete re ac tive oxy gen (su per ox ide) de fen sively in “ox


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ida tive bursts” at sites of pathogen col o niza tion (Lamb and Dixon, 1997). ROS pro duc tion as a re sult of pathogen col o niza tion is known to have a di rect ef fect in killing mi cro bial pathogens (Lamb and Dixon, 1997). This model pro poses that bac te ria are at least weakly path o genic to seedlings. The in va sion of nu clei by bac te ria and the po ten tial for neg a tive ef fects on host nu cleic acids seems con sis tent with this idea. Cur rent re search sug gests that mi cro bivory in vas cu lar plant seedlings may func tion to sup ple ment with nu tri ents of all types needed for growth and de vel op ment. This hy poth e sis de mands that we view plants as mixotrophs. In sup port of this idea, there are an in creas ing num ber of stud ies that sug gest that green plants are in fact mixotrophic, si mul ta ne ously au totroph and het erotroph. Sev eral species of Or chi daceae and Er i caceae have been shown to re ceive car bon from my c or rhizal fungi (Tedersoo et al., 2007; Selosse and Roy, 2008). There is ev i dence that plants se crete pro teases and ab sorb and de grade or ganic forms of ni tro gen, in clud ing amino acids, oligopep tides, and pro teins (Matsubayashi and Sakagami, 1996; Godlewski and Adamczyk, 2007; Kamarova et al., 2008; Jamtgard et al., 2008; PaungfooLonhienne et al., 2008; Hill et al., 2011) and that plants may con sume mi crobes (PaungfooLonhienne et al., 2010a, b; White et al., 2012, 2017). From an eco log i cal per spec tive it is also ap par ent that in some soils in arc tic, alpine, and taiga ecosys tems the an nual plant de mand for ni tro gen far ex ceeds the sup ply of in or ganic ni tro gen in the soil (Kielland, 1994; Näsholm et al., 2009) and thus or ganic forms of ni tro gen are likely used, or they are con verted to in or ganic forms to sup port plant growth. Fur ther, there is the phe nom e non of car nivory in plants, in clud ing venus fly traps, pitcher plants, and sun dews that evolved to cap ture and de grade in sects and small an i mals (Chia et al., 2004; Galek et al., 1990). These plants have evolved to cap ture and de grade in sects, a rel a tively com plex nu tri ent source. For plants, lysing bac te ria pro vides a sim ple nu tri ent source: Bac te r ial walls are thin and they are eas ily degrad able rich sources of nu tri ents. It seems rea son able to hy poth e size that plants ob tain some nu tri ents through mi cro bivory. One of the func tions of the plant mi cro biome may be to pro vide nu tri ents to plants. Pre cisely how im por tant are the nu tri ents de rived from mi cro bivory to fuel plant de vel op ment has rarely been es ti mated, but likely de pends on the plant species and growth stage of the plant. White et al. (2015) con ducted an ex per i ment to es ti mate rhi zobac te r ial con tri bu tion to grass seedling nu tri ent up take. In this ex per i ment, White et al. (2015) la beled bac te ria with iso topic 15N, ex tracted their


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pro teins, and in cor po rated the la beled pro teins into agarose. Tall fes cue (F. arun d i naceae) seedlings with and with out their na tive seed bac te ria were grown on the la beled pro teins, then shoots were as sessed for 15N con tent. Seedlings bear ing the na tive en do phytic rhi zobac te ria con tained ap prox i mately 30% more of the la beled ni tro gen than those that lacked the seedtrans mit ted rhi zobac te ria. This ex per i ment sug gests that as much as 30% of the nu tri ents ob tained by plants could come from mi cro bivory or rhi zophagy. The un cer tainty in this study and the fu ture need is to de ter mine how much of the nu tri ents ab sorbed by plants come di rectly from degra da tion of the bac te ria, and how much comes from ac tiv i ties of the liv ing bac te ria on root sur faces or sur round ing roots in lib er a tion of nu tri ents that may then be ab sorbed by roots. Plant phloemfeed ing in sects of or der Ho moptera have been shown to pos sess en dosym bi otic mi crobes (Pro teobac te ria) within their bod ies. These Pro teobac te ria are in tra cel lu lar “bac te ri o cytes” and they mul ti ply and de grade in time to pro vide pro teins and other nu tri ents for the in sects. Mul ti ple species of Pro teobac te ria are some times pre sent within in sects where they are re ferred to as ‘en dosym bi otic sys tems’ (Koga et al., 2013; Sloan and Moran, 2012). Sim i larly, we hy poth e size that the bac te ria in plant seedlings con sti tute “nu tri tional en dosym bi otic sys tems” of plants that are used as sources of sup ple men tal nu tri ents at times and in cir cum stances when suf fi cient nu tri ents can not be ex tracted from soils.

9.11. THE “RHIZOPHAGY CYCLE” OR “RHIZOPHAGY SYMBIOSIS”

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Re cent ex per i ments have sug gested that plants carry on and within their seeds small com mu ni ties of bac te ria that func tion in a rhi zophagy cy cle (White et al., 2017; Irizarry and White, 2017). In the rhi zophagy cy cle, plants ob tain nu tri ents from bac te ria that al ter nate be tween an in tra cel lu lar/ en do phytic phase and a freeliv ing soil phase (Verma et al. 2017a, b; Prieto et al., 2017; White et al., 2018). Bac te ria ac quire soil nu tri ents in the freeliv ing soil phase; nu tri ents are ex tracted from bac te ria ox ida tively in the in tra cel lu lar/ en do phytic phase (Verma and White, 2018). In the rhi zophagy cy cle plants ma nip u late sym bi otic bac te ria—us ing them as trans porters of soil nu tri ents—then in duce them to en ter into root cell periplas mic spaces at the meris tem tip (White et al. 2017; White et al., 2018); here they ex tract nu tri ents from bac te ria through ox i da tion/


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9.12. CONCLUSIONS

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egra da tion and fi nally plants de posit the sur viv ing bac te ria, ex hausted d of their nu tri ents back into the rhi zos phere, ex it ing from the tips of elon gat ing root hairs (White et al., 2017). The rhi zophagy cy cle could re sult in mo bi liza tion of many nu tri ents (or ganic and in or ganic) from soils by bac te ria and re sult in in creased nu tri ent ac qui si tion by plants (Prieto et al., 2017). Con sid er ing the close and con sis tent as so ci a tion of bac te ria with plants as en do phytes, it is rea son able that plants would de velop ways to ex tract nu tri ents from sym bi otic bac te ria (White et al., 2018).

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Pre vi ous re search (e.g., PaungfooLonhienne et al., 2010a, b; White et al., 2012, 2017) and our seedling sur vey (Table 9.1) sug gest that rhi zophagy is wide spread in vas cu lar plants. It is un known whether some plants rely more heav ily than oth ers on rhi zophagy to ob tain nu tri ents. Large dif fer ences be tween species and cul ti vars were ob served in the amounts of ox i diz ing bac te ria ev i dent in seedling roots (Table 9.1). Dif fer ences were no table in bac te r ial con tent of seedling roots of mod ern white corn, where in tra cel lu lar bac te ria were not ob served, and trop i cal corn, where roots con tained abun dant in tra cel lu lar bac te ria. These dif fer ences could re flect pres ence or ab sence of seedvec tored sym bi otic bac te ria that par tic i pate in rhi zophagy sym bio sis. Pres ence of bac te r ial en do phytes that par tic i pate in rhi zophagy sym bio sis could ex plain why trop i cal corn does not re quire high fer til izer in puts while mod ern hy brid corn de pends on fer til izer in puts. Crop plants such as Moringa oleifera and egusi melon (Cit rul lus colo cyn this) that are high in pro teins and other nu tri ents (Juliani et al., 2010; Akubundu et al., 1982) could rely on bac te ria in volved in rhi zophagy sym bio sis to scav enge nu tri ents in soils and carry them back to plants where they may be ex tracted ox ida tively and ab sorbed into root cells. Be cause the rhi zophagy cy cle in volves bac te ria that are ox i dized within roots, plants that are ac tively in volved in a rhi zophagy sym bio sis not only ac quire nu tri ents but they also may upreg u late genes for ox ida tive stress re sis tance and con se quently are re sis tant to abi otic and bi otic stresses (White and Torres, 2010). We are only just be gin ning to rec og nize rhi zophagy sym bio sis and many top ics need to be ad dressed. For ex am ple, some ques tions in clude: Are there nu tri ent spe cial ists among the sym bi otic bac te ria that are bet ter a trans port ing to plants par tic u lar nu tri ents? How much of the nu tri ents ab sorbed by plants


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comes di rectly from in ter nal mi crobe degra da tion; how much comes from ac tiv i ties of the mi crobes in the rhi zos phere in lib er at ing soil nu tri ents that are ab sorbed by roots? Are there bac te ria that cause plants to ex press ox ida tive stress tol er ance but do not carry nu tri ents? Can we move the seedvec tored sym bi otic bac te ria be tween species of plants (Verma and White, 2018) or cul ti vars and im prove rhi zophagy cy cle ac tiv ity in crop plants where na tive sym bi otic bac te ria have been lost? Pre cisely what nu tri ents do plants ob tain from rhi zophagy sym bio sis? Does the plant ab sorb into root cells par tially de graded or ganic mol e cules—or are all or ganic mol e cules com pletely ox i dized prior to ab sorp tion? Do bac te ria that col o nize root meris tems also col o nize shoot meris tems and thus be come dis trib uted to all parts of plants? How do plants in duce sym bi otic bac te ria to en ter cells at the root meris tem? What are the “sig nal” mol e cules that pass be tween plant and bac terium dur ing the sym bi otic in ter ac tions? What genes do hosts ex press dur ing rhi zophagy ac tiv i ties? What root cells (su per fi cial lay ers on root sur face or cells deep in the in te rior of the root axis) are in volved in the rhi zophagy process? Much ad di tional re search will be needed be fore we will fully un der stand the rhi zophagy process or its ram i fi ca tions for crop pro duc tion. Re gard less of what is still un known, it is in creas ingly clear that rhi zophagy sym bio sis may rep re sent an im por tant nu tri tional sym bio sis that func tions in many or all plants to pro vide nu tri ents. Rhi zophagy sym bio sis may rep re sent a prim i tive but fun da men tal process for nu tri ent ac qui si tion func tion ing in seed less vas cu lar plants such as ferns as well as seed plants. Un der stand ing how rhi zophagy sym bio sis func tions could lead to new ways to cul ti vate crops with out re liance on ex ces sive agro chem i cal ap pli ca tions. Fi nally, learn ing how to ma nip u late rhi zophagy sym bio sis could also re sult in new tech nolo gies for re duc ing growth of weedy or in va sive plant species by in hibit ing the sym bio sis.

ACKNOWLEDGMENTS

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The au thors ac knowl edge the De part ment of Plant Bi ol ogy, Rut gers Uni ver sity, NJ for re search fa cil i ties and fi nan cial sup port. SKV is thank ful to UGC, In dia for pro vid ing a Ra man Post Doc toral fel low ship (No.F 511/ 2016 IC) for the year (2016–17) to con duct re search in USA. SKV is grate ful to the Head and Co or di na tor CAS, FIST of Botany, B.H.U., Varanasi, In dia for pro vid ing the leave to pur sue re search on en do phytes. The au thors are also grate ful for sup port from the John E. and Christina C. Craig head Foun da tion, USDANIFA Mul ti state Pro ject W3147, and the New Jer sey Agri cul tural Ex per i ment Sta tion. Funds for part of this work were from Co op er a tive Ecosys tems Stud ies Unit CESU G16AC00433 be tween Rut gers Uni ver sity and the US Ge o log i cal Sur vey for con trol of in va sive P. aus tralis. We are grate ful to Ilya Raskin, Car rie Wa ter man, and Al bert Ayeni for seeds of moringa and egusi melon used in this study.


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