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Phytohormonal cross-talk modulate Bipolaris sorokiniana (Scc.)interaction with Zea mays

Besides acting as growth inducing molecule, Gibberellin (GA3) also confers the compatibility of microbial interactions with host. We inoculated 11 days old Z. mays seedlings grown under hydroponic conditions and high GA3 levels with Bipolaris sorokiniana (BIPOL) at the spore density (SD) of OD0.6. The high level of GA3 negatively affected the growth of the seedlings, accompanied by the high level of stress deducing secondary metabolites (proline, total flavanoids, phenylpropanoids, and glucosinolides). Moreover, high level of GA3 produced a hypersensitive response (HR) in the seedlings. The HR developed cross talks with IAA and trans-zeatins and triggered higher production of hypersensitive inducing biomolecules. The other HR co-related biological processes were demonstrated by high phytoalexins level and high protease activities. Such activities ultimately inhibited the colonization of BIPOL on the roots of maize seedlings. The products of the genes expressed at high GA3 also conferred the deterrence of BIPOL colonization at SD = OD0.6. Intriguingly, when we inhibited GA3 biosynthesis in the seedlings with aerially sprayed uniconizole, prior to BIPOL treatment, the BIPOL colonized and subsequently promoted the seedling growth. This low level of GA3 after BIPOL treatment checked the high level of secondary metabolites and hypersensitivity inducing molecules. The results, thus suggested that the aforementioned processes only happened in the BIPOL at SD (OD0.6), whereas the SD at lower levels (OD0.2 or OD0.4) neither promoted the growth of uniconizole pre-treated seedlings nor produced HR in control seedlings of maize plant.


Introduction
Prior to inoculation, the spore was washed by centrifugating at 10 5 xg at 10 ºC for 30 min in 126 sterile distilled water trice (Huttenlocher et al., 2019). Optical density (OD) of the washed 127 spore's suspension was measured at 600 nm and adjusted to OD 0.2 or OD 0.4 or OD 0.6 with the 128 help of sterilized distilled water. We used germinated spore to inoculate the plant to 129 accelerate the colonization potential of B. sorokiniana (Selvakumar et al., 2018).

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Determination of fungal root colonization 131 Root segments of seedlings of the fungal treatment were kept on a Petri plate containing PDA 132 media and then incubated at 25 ºC. To calculate fungal colonization frequency, the relative 133 number of the root segments occupied by the fungus was observed. 135 For phytochemical analyses, fresh leaves were quickly frozen in liquid nitrogen and ground 136 to obtain fine powder in a mortar and pestle. Absolute methanol (200 mL) was then added to 137 the fine powder (2 gm) and the solution was transferred to the soxhlet apparatus for the extraction of phytochemicals (Dhawan and Gupta, 2017). The obtained leaf extract (LE) was 139 first filtered and the filtrate was concentrated in the rotary evaporatory (Wahyuningsih et al.,140 2017). Moreover, root exudates (RE) was obtained from filtered hypdroponic culture of the 141 root seedlings and concentrated in the rotary evaporatory.

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Secondary metabolite determination 143 Colorimetric method was used for the evaluation of total flavonoids and proline contents. 144 Samples (1 mL) of LE and RE were prepared as mentioned above, mixed in 3 % sulfo-145 salicylic acid (4 mL) and centrifuged for 5 mins at 6077 rcf. After centrifugation, the 2 mL of 146 ninhydrin reagent was added to the samples and shaken vigorously. Acid ninhydrin reagent 147 was prepared by adding 1.25 g of ninhydrin in pure glacial acetic acid (30 mL) and 20 mL of 148 phosphoric acid (6 M) and mixed. The reaction mixture was heated for 1 hour at 100 ºC and 149 the pellet found was disappeared in toluene (4 mL) (Lee et al., 2018). OD of the samples 150 were monitored using UV/Vis spectrophotometer (Lambda 1050) at 520 nm. Similarly, for 151 determination of total flavonoid contents, 0.5 mL of LE and RE samples were added into 10 152 % potassium acetate (100 µL), 10 % aluminium chloride (100 µL), 70 % ethanol (4.3 mL).

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Freshly taken leaf or root section (2 gm) was homogenised by centrifugation at 5000 g, for 30 184 min. The cell pellet was suspended in 1 mL extraction buffer (40 % methanol, 0.1 % formic 185 acid, 40 % acetonitrile, and 19.9 % distilled water) and vortexed for 30 sec. The sample was 186 incubated for 30 min. after incubation, lysing was done in by using non-contact ultra-187 sonication (UCD-200, Belgium) (Jenal et al., 2017). The samples were subjected to HPLC-the standards of the pure c-di-GMP and cAMP levels.

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After removing root or leaf tissues, samples were acidified by adding 50 µL HCL (1.6 M).

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Phase separation was done through mixing ethyl acetate (2 mL) and then dried into nitrogen tubes. The samples were concentrated using N 2 flow (10 bar) and later heated at 42 ○ C. The 237 dried samples (2 mg) was then mixed in 2 mL of methanol in water (95.9 %). The samples 238 were filtered by spin-X centrifuge tube at 10,000 x g.

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The samples (phytoalexins extracted) were then exposed to HPLC coupled MS by 240 injecting 20 µl sample into HPLC (Ultimate 3000) armed with a Reverse-phase column 241 (Acclaim120 C18) at 35 ○ C with flow rate 0.2 mL/min. The mobile phase used was prepared   Table S1: List of primers used for the expression of 9 markers gene in maize seedlings 288

S. No. Genes in A. thaliana
Homologue of Maize Gene (ID)  (Figure 2A-2B). Recorded data revealed that growth was promoted once GA 3 was 297 inhibited after BIPOL inoculation at SD (OD 0.6 ) ( Figure 1C & 1F). However, the growth 298 was found to be very low at uniconizole treatment till 72 hour, while later restored ( Figure   299 1A & 1D) . Surprisingly, when we challenged the host with BIPOL alone at SD (OD 0.6 ), the 300 growth of seedlings became highly compromised ( Figure 1B & 1E). Moreover, when we 301 treated the seedlings with BIPOL SD (OD 0.2 or OD 0.4 ), the seedlings did not respond to both 302 SDs ( Figure S1A & S1C). Similarly, the both SDs also could not promote growth at 303 uniconizole pre-treated seedlings when maize seedlings were at stress till 72 h (Figure S1B-304 S1D).

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Cell wall integrity is very essential for the host under growth promoting condition 306 (Mirabet et al., 2018). We determined the integrity by Electrolytic leakage (EL) as high EL 307 value refers to host cell wall stability (Mihailova et al., 2018). As expected, EL was low in 308 U-B seedlings at SD (OD 0.6 ) ( Figure 1I). However, the uniconizole pre-treated seedlings of 309 maize had high EL till 72 h which later became same as control ( Figure 1G). As opposed,

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EL was very high in BIPOL treated seedlings of maize at SD (OD 0.6 ) ( Figure 1H). Similarly, 311 there found no change observed in EL at SD (OD 0.2 or OD 0.4 ) in control or with pre-treatment 312 of uniconizole to maize seedlings (Figure S1E-S1F).

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The interacting microbe alters the certain specific host hormonal level to successfully 315 colonize in the host tissues (Chagas et al., 2018). Such altered hormone level produced a 316 cross talk with other hormones which in turn produce a HR in host (Bürger and Chory, 2019).

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In case of BIPOL interaction with host, we observed essential hormonal cross talks of GA 3 ,

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Oxidase activity is induced upon the elevated stress condition on plant (Joshi et al., . As expected, uniconizole treatment to maize seedlings increased oxidase activity till 371 72 hour which later became normal as control. As opposed to this, the oxidase activity was 372 low in U-B seedlings of maize at SD (OD 0.6 ) ( Figure 4A-4E). Interestingly, the BIPOL 373 inoculation SD (OD 0.6 ) also increased oxidase activity till 120 hours in maize seedlings 374 ( Figure 4A-4E). Additionally, the BIPOL inoculation at SD (OD 02 or OD 0.4 ) could not 375 induced oxidase activity (Figure S4E-S4F). Similarly, both SD (OD 02 or OD 0.4 ) also could 376 not reduce oxidase activity in uniconizole treated seedlings of maize (Figure S4E-S4F). streaming to promote the microbe interaction were also upregulated ( Figure 9B). 441 We selected the 9 marker genes and analyzed its expression during qRT-PCR in 11 days olf 442 maize seedling under treatments. Results deducted from qRT-PCR indicated that expression 443 of WII2 (Zm00001d002065), TRX5 (Zm00001d002690) and PTF1 (Zm00001d045046) was 444 highly reduced at U-B seedlings, while the same were upregulated at BIPOL treatment to 445 maize seedlings (Figure 10A-10E). No significance difference was found in uniconizole 446 treated seedlings of maize compared to control (Figure 10A-10E). As opposed to these 447 expressions, the expression of DPMS2 (Zm00001d034682), TAT3 (Zm00001d053107) and 448 WRKY15 (Zm00001d036542) was high at U-B seedlings while the same was low at BIPOL 449 treatment to maize seedlings (Figure 10F-10J). No significance difference was found in 450 uniconizole treatment compared to control (Figure 10F-10J). 451 However, the expression of GID1 (Zm00001d010308), RGA1 (Zm00001d041362) and 452 ga20ox2 (Zm00001d007894) was remained unaffected in uniconizole treatment to maize 453 seedlings ( Figure 10K-10O). As opposed, the expression of GID1 (Zm00001d010308) and 454 ga20ox2 (Zm00001d007894) was very high while RGA1 (Zm00001d041362) was low in 455 BIPOL treatment (Figure 10K-10O). The U-B seedlings of maize followed the exact 456 opposite trend in the expression of these three genes compared to BIPOL treatment as the 457 expression of GID1 (Zm00001d010308) and ga20ox2 (Zm00001d007894) was very low 458 while the expression of RGA1 (Zm00001d041362) was high (Figure 10K-10O).

Root colonization of BIPOL under high GA 3 concentration 460
Fungal colonization was noted very low for BIPOL treated seedlings at SD (OD 0.6 ), whereas 461 it was relatively high in U-B seedlings compared to control seedlings of maize at SD (OD 0.6 ) 462 ( Figure 11A). However, there was found no change in percent colonization of BIPOL at SD 463 (OD 0.2 or OD 0.4 ) in both BIPOL and U-B treated seedlings of maize ( Figure 11B). Determination of RGR (relative growth rate), NAR (net assimilation rate), and EC 472 (electrolytic content leakage) in the 11 days old maize seedlings exposed to different Determination of Oxidase, Catalase enzymatic activities, NAD + (cofactor) and FAD +

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(cofactor) in the 11 days old maize seedlings exposed to different treatments including C  Uniconizole treated seedlings nor control seedlings.

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This deducted that the two BIPOL SD (OD 0.2 or OD 0.4 )) treatment to maize seedlings are 680 inert, neither growth promoting and virulent. The same pattern was also followed by HR (control seedlings treated with BIPOL SD (OD 0.6 )), the BIPOL colonization was hindered.

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This could be due to the high level of GA 3 which developed cross talks with IAA and 710 Tranzeatin, thus decreasing its production in host.

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Beside growth inducing molecules, GA 3 also indicates the compatibility of host-micorbial 713 interaction. Inoculation of BIPOL majorly caused GA 3 hypersentivity at a SD (OD 0.6 ) at 600 714 nm. this hypersensitivity interfered the optimal level of IAA and Trans-zeatin in the host, 715 thereby demoting growth of the seedlings. Once the GA 3 was inhibited using uniconizole 716 treatment BIPOL SD (OD 0.6 ) colonized on the seedlings root and resumed its growth 717 promoting acitivity. This supported our hypothesis that GA 3 hypersensitivity hinders the 718 interaction of BIPOL with Z. mays under its cross talks with IAA and trans-zeatin.