Suberin Biopolymer in Rice Root Exodermis Reinforces Preformed Barrier Against Meloidogyne graminicola Infection

doi:10.1016/j.rsci.2021.04.008

Abstract

Abstract:

Exploration of novel genetic resources against root-knot nematode (RKN) is necessary to strengthen the resistance breeding program in cultivated rice, and investigations on the role of genotype-specific root anatomy in conferring a structural barrier against nematode invasion are largely underexplored. Here, we reported a highly-resistant rice germplasm Phule Radha that conferred remarkably lower RKN parasitic fitness in terms of reduced penetration and delayed development and reproduction when compared with susceptible cultivar PB1121. Using histological and biochemical analyses, we demonstrated that an enhanced suberin deposition in the exodermal root tip tissue of Phule Radha compared to PB1121 can effectively form a penetrative barrier against RKN infection, and this preformed barrier in the control tissue did not necessarily alter to a greater extent when challenged with RKN stress. Using qRT-PCR analysis, we showed that a number of suberin biosynthesis genes were greatly expressed in the exodermis of Phule Radha compared to PB1121. In sum, the present study established the role of rice exodermal barrier system in defense against an important soil-borne pathogen.

Key words: penetration, suberin lamellae, root-knot nematode, rice root exodermis

Singh Divya, K. Dutta Tushar, N. Shivakumara Tagginahalli, Dash Manoranjan, Bollinedi Haritha, Rao Uma. Suberin Biopolymer in Rice Root Exodermis Reinforces Preformed Barrier Against Meloidogyne graminicola Infection[J]. Rice Science, 2021, 28(3): 301-312.

Figures/Tables 12

Fig. S1. Hypothesis of present study schematically represented. In low suberin containing rice root (indicated by dotted perpendicular red line), J2s of M. graminicola can penetrate the root at elongation zone, migrate to undifferentiated meristematic tissue and reach vascular cylinder by making a U-turn bypassing the differentiated endodermis. By contrast, in high suberin containing rice root (indicated by solid perpendicular red line), J2s of M. graminicola cannot cross the exodermis barrier and even if penetrated GCs induced in the vascular tissue become nonfunctional. Figures are not drawn to scale. N, Nematode; F, Adult female; E, Egg mass; GC, Giant cell.

Fig. S2. Screening of rice varieties for nematode resistance.Relative numbers of gall, J2, J3/J4, egg mass and eggs/egg mass of M. graminicola in different varieties of rice (PB1121, Suraksha, Vandana, EK70, Khalibagh and Phule Radha) at 16 d after inoculation. Different lowercase letters within any parameter are significantly different at P < 0.01 by the Tukey’s HSD test. Error bars indicate standard error of mean (n = 6). Inoculum level was 30 J2s/plant.J2, J3 and J4 refer to the second, third and fourth stage juveniles, respectively.

Fig. 1. Meloidogyne graminicola infected plantlets of PB1121 and Phule Radha in Petri plates containing PF-127 medium at 16 d after inoculation. A, Photographs show comparatively greater galling intensity and larger sized galls (indicated by arrows in bottom panel magnified view) in PB1121 than negligible and smaller sized galls in Phule Radha. Scale bars, 1 cm.B, Relative numbers of galls, egg masses, eggs/egg mass and multiplication factor (MF) ratio in PB1121 and Phule Radha. Inoculum level is 30 J2s/plant. J2, The second stage juveniles; J3/J4, The third/fourth stage juveniles. Data represent Mean ± SE (n = 3). Asterisks indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.

Table S1. Comparison of M. graminicola invasion, development and reproduction in PB1121 and Phule Radha.

Life cycle stage	Days after inoculation (d)	Number of nematodes/root system
Life cycle stage	Days after inoculation (d)	PB1121	Phule Radha
J2	1	25.66 ± 1.20 e	5.33 ± 0.33 b
	2	32.33 ± 2.67 f	9.66 ± 0.67 c
	3	34.00 ± 2.88 fg	10.33 ± 0.58 d
	4	35.50 ± 2.76 g	11.25 ± 1.18 d
	5	8.80 ± 1.53 c	5.50 ± 0.58 b
	7	5.00 ± 0.88 b	2.33 ± 0.33 a
J3/J4	5	27.00 ± 1.15 d	5.00 ± 0.67 ab
	7	26.66 ± 1.45 d	6.25 ± 1.20 b
	10	8.00 ± 0.88 c	4.66 ± 0.66 a
	12	0.00 ± 0.00 a	3.33 ± 0.33 a
Female	10	24.00 ± 1.67 b	0.00 ± 0.00 a
	12	27.50 ± 2.57 c	3.66 ± 0.33 a
	16	33.00 ± 1.76 d	4.66 ± 0.66 a
Egg mass	16	33.00 ± 1.76 b	4.66 ± 0.66 a
Eggs/egg mass	16	152.80 ± 8.76 b	10.66 ± 1.58 a
Multiplication factor ratio		50.42 ± 6.17 b	0.49 ± 0.13 a

Fig. 2. Comparative life cycle progression of M. graminicola in PB1121 and Phule Radha. Typical hook-like gall formation at the root tip was evident from 2 DAI onward in PB1121. By contrast, Phule Radha supported fewer galls because of the lower J2 penetration and delayed development of invading J2s even at 12 and 16 DAI. Nematodes were stained with acid fuchsin. White arrows indicate root galls. Scale bars, 200 µm.DAI, Days after inoculation; J2, J3 and J4, The second, third and fourth stage juveniles, respectively; E, Eggs; F, Female.

Fig. 3. Comparative attraction to and penetration of PB1121 and Phule Radha root tips by M. graminicola. A, Attraction of the second stage juveniles (J2s) towards rice root tips in the PF-127 medium at 2, 4, 6, 8 and 10 h after inoculation. Data represent Mean ± SE (n = 3) and bars with unshared letters (within identical time point) indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.B, Photomicrographs showing attraction of J2s to the root tips of PB1121 and Phule Radha at 8 h after inoculation. Head of the young female is localized among the multinucleate giant cells with densely stained cytoplasm at the vascular cylinder of PB1121. By contrast, nematode head surrounded by the giant cells are devoid of cytoplasm at the vascular cylinder of Phule Radha. C, Penetration of J2s in rice root tips at 24, 48 and 72 h after inoculation. Data represent Mean ± SE (n = 3) and bars with unshared letters (within identical time point) indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.D, Cross-sections of rice roots infected by M. graminicola at 7 d after inoculation. n, Nematode; *, Giant cell.

Fig. 4. Chemotaxis of M. graminicola towards root exudates of PB1121 and Phule Radha. A, Assay was conducted in a 50 mm × 10 mm Petri dish containing PF-127 in central area and agar in the peripheral area. Wells containing 10 µL of root exudate (sterile water as control) were 1.5 cm distant from the nematode inoculation point (100 J2s applied). J2s, The second stage juveniles.B, A close-up view of the assay plate shows that after 1 h of inoculation, J2s were accumulated near the point source at agar-PF-127 interface when exposed to root exudates of PB1121. C, Chemotaxis index of nematodes towards rice root exudates. Marigold was used as a negative control. Values indicate attraction (positive index) or repulsion (negative index). Data represent Mean ± SE (n = 3) and bars with unshared letters indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.

Fig. 5. Suberin content in root tips of PB1121 and Phule Radha. Aliphatic and aromatic suberin released from the exodermis of control and nematode infected [5-day-old plantlets were inoculated with 100 J2s (the second stage juveniles) of M. graminicola in the PF-127 medium and plantlets harvested at 2 d after inoculation] roots after transesterification with BF3-methanol. Total aliphatic suberin amount and monomer compositions were performed via gas chromatography- mass spectrometry analysis. Bars represent Mean ± SE (n = 3) and bars with unshared letters (within each substance class) indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.

Fig. 6. Comparative suberin lamellae deposition in roots of PB1121 and Phule Radha. Cross sections of the control and nematode-infected rice roots stained with Fluorol Yellow 088. Intense yellowish green fluorescence (indicated by white arrowheads) in the suberized exodermis of Phule Radha is evident. Blue fluorescence indicates auto fluorescence. Sections of both genotypes were taken at the identical distance from root tip (about 1 cm from root apex). Scale bars, 50 µm. ep, Epidermis; ex, Exodermis/hypodermis; sc, Sclerenchyma; co, Cortex.

Fig. 7. Comparative Casparian strip (CS) deposition in roots of PB1121 and Phule Radha. Cross sections were obtained from 5-day-old control roots and stained with berberine hemisulfate and aniline blue. Intense green fluorescence (indicated by white arrows) in the CS lining of Phule Radha exodermis (top panel) and endodermis (bottom panel) is evident. Berberine causes intense xylem (X) fluorescence. Aniline blue counterstains berberine- induced fluorescence of lignin and reveals endodermal CS. Sections of both genotypes were taken at the identical distance from root tips (about 1 cm from root apex). Scale bars, 20 µm. ep, Epidermis; ex, Exodermis/hypodermis; sc, Sclerenchyma; co, Cortex.

Fig. 8. Relative expression levels of suberin biosynthesis genes in root tip exodermis of PB1121 and Phule Radha. Five-day-old plantlets were inoculated with 100 J2s (the second stage juveniles) of M. graminicola in the PF-127 medium and harvested at 2 d after inoculation. Uninfected roots served as the control. qRT-PCR data are expressed as the fold change in expression from three biological and three technical replicates each containing a pool of 10 plants. Gene expression levels were normalized using the internal reference gene of O. sativa, i.e. 18S rRNA. Data represent Mean ± SE and bars with unshared letters (within each gene) indicate significant difference at P < 0.01 by the post-hoc Tukey’s test.

Table S2. List of oligonucleotides employed for qRT-PCR analysis of suberin biosynthesis genes in rice.

Gene name	Forward primer (5′-3′)	Reverse primer (5′-3′)
CYP86A1	CTCTAAACTTCGACGAGCTG	CCCACTGAGTAGATGGAGTA
CYP86B1	CCGGTTTCATAGCGAAACTAC	GAAGGTGCAAATCCCTCTT
FAR1	CAAGTACTCCTTCGTCATGC	GTCCCAGTCGATGTTCTTG
GPAT5	CCAACTACGTGCAGAGGATAC	GAAGCCGAGAACCTCCTTG
KCS2	CAACTGCAGCCTCTTCAAC	CAGTTGAGCGTGATGTTCTC
ASFT	CTCTACTACCTCTCCAACCT	AGGGTAGTAGTGGACAAGGA
ABCG2	AGGTGTTTCAGAGAGAGAGG	AGGACGAAGAAGAGGTAGTG
GDSL	CTTCTACTTCGTGGCAATCG	CGCTGAAGAGGAGGAAGTA
LACS4	CATGCGGTGGTTCTATTCTG	CAACATATGGGCACACACTC
HOTHEAD	GTGGAATTGGACCCAGAAAG	CGTCGGTTATTCCAACAGTC
MYB107	CTCATCGCCTACATCCAGAA	CTTGATGATGGTGTCCTCCT
18S rRNA	CGCGCAAATTACCCAATCCTGACA	TCCCGAAGGCCAACGTAAATAGGA

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